CN102187224A - Methods for detecting a mycobacterium tuberculosis infection - Google Patents

Abstract

Methods for detecting an infection with Mycobacterium tuberculosis (Mtb) in a subject are disclosed, wherein the subject is a child, a subject with a latent Mycobacterium tuberculosis infection. Method are also disclose for detecting an extra-pulmonary Mycobacterium tuberculosis infection in a subject. The methods include detecting the presence of CD8+T cells that specifically recognize an Mtb polypeptide. The methods include in vitro assays for detecting the presence of CD8+T cells in a biological sample.

Description

Be used to detect the method for m tuberculosis infection

Prioity claim

The present invention requires the U.S. Provisional Application No.61/099 that submitted on September 28th, 2008,162 right of priority, described provisional application to draw at this to be reference.

The statement of government-funded

The present invention finishes under the support of U.S. government according to (National Institutes of Heath) subsidy AI054474 of NIH and AI070022; U.S. government has certain right in the present invention.The present invention also finishes under the support of Department of Veterans Affairs (Department of Veterans Affairs).

Technical field

The application relates to diagnostic field, relate in particular to be used for object particularly children detect method that Much's bacillus (Mycobacterium tuberculsosis) (Mtb) infects and/or the method that is used to diagnose latent infection.

Related subject

The U.S. Provisional Application No.60/782 that the application relates on March 14th, 2006 and submits to, the theme of the PCT that submitted on March 14th, 364 and 2007 application No.PCT/US2007/006534, said two devices draw at this and are reference.

Background technology

Mycobacterium is the aerobic interior bacterium living beings generic of cell, and it is in the endosome compartment (endosomal compartments) of survival at monocyte and macrophage behind the infection host.Human mycobacterium disease comprises tuberculosis ((M.tuberculosis) causes by Much's bacillus), leprosy ((M.leprae) causes by the leprosy mycobacterium), Bairnsdale canker ((M.ulcerans) causes by mycobacterium buruli) and by Mycobacterium marinum (M.marinum), mycobacterium kansasii (M.kansasii), scrofula mycobacterium (M.scrofulaceum), Suhl adds branch bacillus (M.szulgai), mycobacterium xenopi (M.xenopi), mycobacterium fortutitum (M.fortuitum), mycobacterium chelonei (M.chelonei), the various infection that mycobacterium haemophilum (M.haemophilum) and Mycobacterium intracellulare (M.intracellulare) cause are (referring to Wolinsky, E. the 37th chapter in " microbiology: comprise immunology and molecular genetics " (the Microbiology:Including Immunology and Molecular Genetics) third edition, Harper ﹠amp; Row, Philadelphia, 1980).

/ 3rd of a world population has Much's bacillus, and is in the risk that tuberculosis (TB) takes place.Young child has out-of-proportion tuberculosis (TB) morbidity burden.In case infect, children not only than the adult to TB susceptible more, and serious disease form more likely takes place.Specifically, after infection, surpass 90% immune adult and will set up TB asymptomatic, that hide and infect (LTBI), it has the risk of reactivation disease in lifetime of 5-10%.Yet in most of infants, former Mtb infects and will develop into activity TB, and in the object of suffering from activity TB of significant proportion, disease will develop into more serious form (for example grain graininess TB).Except the TB neurological susceptibility was increased, the timely diagnosis in children was complicated because of the following fact, and the children that promptly suffer from the primary infection of carrying out property seldom present the acid-fast bacilli phlegm smear positive, and this is usually can be observed in adult lung reactivation disease.Early detection is essential, because make progress in diagnosis timing period disease.

In immunocompromised patient, tuberculosis to be increasing near logarithm speed, and the multi-drug resistance bacterial strain occurred.In addition, the present main killer who has become immunosuppressant AIDS patient of mycobacterium strain (for example mycobacterium avium (M.avium)) who was considered to the avirulence bacterial strain in the past.In addition, present mycobacterium vaccine good inadequately (the BCG vaccine that for example is used for Much's bacillus) maybe can not obtain (for example for the leprosy mycobacterium) (Kaufmann, S., Microbiol.Sci.4:324-328,1987; " lasting challenge lungy " (U.S.Congress of OTA of US Congress, Office of Technology Assessment, The Continuing Challenge of Tuberculosis), the 62-67 page or leaf, OTA-H-574, united states government printing office (U.S.Government Printing Office), Washington, D.C., 1993).

Suppress the accurate early diagnosis of effective immunity inoculation of propagation needs lungy and disease.At present, using the immunity inoculation of the bacterium that lives is the most effectual way of inducing protective immunity.The most frequently used mycobacterium that is used for this purpose is Bacille Calmette-Guerin (BCG), and this is the had no pathogenicity bacterial strain of Mycobacterium bovis (Mycobacterium bovis).But the security of BCG and effect are the sources of arguement, and some country for example the U.S. not to the general public immunization campaign.

Diagnosis lungy uses skin test to carry out usually, and it relates to and is exposed to tuberculin PPD (protein purification derivant) in the corium.After injection 48 to 72 hours, T cells with antigenic specificity was replied at the injection site place and is produced measurable scleroma, and it shows and is exposed to antigen of mycobacterium.But the sensitivity and the specificity of this test are undesirable; Individuality with the BCG immunity inoculation can not distinguish with infected individuality.In addition, it is not effective especially in diagnosis children or LTBI.Therefore, in the art, to be used for detecting tuberculosis children, especially for detecting LTBI and being used to the improved diagnostic method of diagnosing TB to infect, have demand.

Summary of the invention

Herein disclosed is the method that is used to diagnose Much's bacillus (Mtb) infection.In certain embodiments, method is used for detecting the tuberculosis infection (LTBI) of hiding and/or detecting the Mtb infection children.In other embodiments, method is used to detect lung and infects outward.Method comprises separation of C D8 ⁺T cell and detection are to the CD8+T cell of target Mtb polypeptide generation specificly-response.Method can comprise the expression of detection cell factor such as but not limited to interferon (IFN)-γ.In certain embodiments, method has been utilized ESAT-6 and/or CFP-10 polypeptide, such as but not limited to detect tuberculosis in children.

In several embodiments, provide the method that is used for detecting Much's bacillus at object.These methods can be used for detecting tuberculosis, comprise pulmonary tuberculosis and/or extrapulmonary tuberculosis.These methods comprise and will contain T cell, for example CD8 from object ⁺The biological sample of T cell contacts with one or more mycobacterium polypeptide or the antigen presenting cell of presenting one or more mycobacterium polypeptide.Described one or more mycobacterium polypeptide can comprise ESAT9 and CFP10 or its antigenic epitopes.Described one or more mycobacterium polypeptide also can comprise following shown amino acid sequence: (a) one of shown amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:39 or SEQ ID NO:61; Or (b) at least 9 to 20 continuous amino acid of shown at least one amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:39 or SEQ ID NO:61, wherein said 9 to 20 continuous amino acid specificitys are in conjunction with the main histocompatibility complex of I class (MHC); One of or the shown amino acid sequence of SEQ ID NO:39-83.Determine whether specific recognition mycobacterium polypeptide of T cell.

In other embodiments, method comprises that also subcutaneous or intradermal administration is in subject's skin with the mycobacterium polypeptide of effective dose.The mycobacterium polypeptide can be ESAT6 or CFP10 or its antigenic epitopes.The mycobacterium polypeptide comprises following shown amino acid sequence: (a) one of shown amino acid sequence of SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ IDNO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:31 or SEQ ID NO:61; Or (b) SEQ ID NO:1, SEQ ID NO:2, SEQID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQID NO:11, SEQ ID NO:12, at least 9 to 20 continuous amino acid of at least one amino acid sequence that SEQ ID NO:39 or SEQ ID NO:61 are shown, one of wherein said 9 to 20 continuous amino acid specificitys are in conjunction with the main histocompatibility complex of I class (MHC), or the shown amino acid sequence of SEQ ID NO:39-83.The existence of the T cell of detection specificity identification mycobacterium polypeptide in object.

Method can also be included in and detect delayed allergy in the object and/or can comprise detection specificity Mtb polypeptide and polynucleotide.Disclosed analysis can be used alone or in combination.M tuberculosis infection can be to hide or movable the infection.

In addition, reagent and the kit that is used for detecting at object mycobacterial infections described.

From the detailed description of several embodiments of carrying out below with reference to accompanying drawing, above-mentioned and other characteristics and advantage will become more obvious.

The accompanying drawing summary

Fig. 1 has shown that use IFN-γ ELISPOT divides dialyse (ex vivo) to measure two figure of human effector cell's frequency.CD8 with the magnetic beads purifying ⁺T cell and DC (20,000/ hole) cultivate in IFN-γ ELISPOT analyzes together, described DC or infected by Mtb that (H37Rv is MOI=50) or with peptide merging thing (every kind of peptide 5 μ g/ml, 15 monomers, overlapping 11 amino acid) pulse of presenting CFP10.Each response T cell colony is tested double under four kinds of different T cell concentrations.In order to determine effector cell's frequency of T cells with antigenic specificity, the average spot number in each hole of the parallel sample of each double is mapped to the response cell number in each hole.Use linear regression analysis to determine the slope of line, the frequency of its expression T cells with antigenic specificity.If the binomial probability of the spot number of experiment and check analysis is significantly different, analyze (the reflecting the existence that sensitized T cell is replied) that be considered to positive.

Fig. 2 is one group and has shown the stripped CD8 to Mtb antigen ⁺T cell frequency infects relevant figure with Mtb.(referring to Fig. 1) as mentioned above is in order to determine the CD8 that exsomatizes ⁺T cell frequency, will with Mtb infect or with homeopeptide merge the thing pulse from body DC and CD8 ⁺T cell incubation together in IFN-γ ELISPOT analyzes.To do not have Mtb to infect the object of sign, the object (lung's tuberculosis that culture confirms) of suffering from the object of LTBI and suffering from activity TB is assessed." Mtb infection " comprises suffering from LTBI and active tuberculosis.When P=＜0.05 (Wilcoxon/Kruskal-Wallis), indicated the P value.

Fig. 3 a is one group to 3d and has shown and define antigentic specificity and the digital picture of HLA restricted (sign of T cell clone D466D6).For result displayed among Fig. 3 a-3c, for antigen and the minimum epi-position of identifying that T cell clone D466D6 is discerned, with T-cell (5000 cells/well) with from body LCL (20,000/ hole) and 5 μ g/ml antigens incubation together.After cultivating 18 hours altogether, assess IFN-γ by ELISPOT.For result displayed among Fig. 3 a, antigen is by presenting known CD4 ⁺The peptide of antigen merges thing and constitutes, and peptide merges thing peptide long by 15 amino acid (aa), that have 11 overlapping amino acids and forms.For result displayed among Fig. 3 b, antigen is made of each 15 amino acid whose CFP10 peptides that lump together component peptide merging thing.For result displayed among Fig. 3 c, antigen is by each nested CFP10 _1-15Peptide (10 amino acid, 9 amino acid or 8 amino acid) constitutes, and is used for the further mapping of epi-position.For result displayed among Fig. 3 d, use with CFP10 _2-10(5 μ g/ml) pulse, express the allelic LCL of HLA (20,000/ hole) with the D466 coupling at one or two allele place as APC, identify restriction allele.After 2 hours, clean cell and with T-cell (500 cells/well) incubation in IFN-γ ELISPOT analyzes.

Fig. 4 has shown the line chart of confirming the minimum epitope mapping of D466D6.In order to confirm minimum epi-position, will carry out pulse with the peptide of indicating concentration from body LCL (20,000/ hole), and cultivate altogether with T-cell (1000 cells/well).After cultivating 18 hours altogether, assess IFN-γ by ELISPOT.The mean value of two parts of replicate determinations of each some expression.

Fig. 5 is the bar chart of the distribution situation (profiling) of one group of immunodominance pattern that has shown CFP10.In order to determine effector cell's frequency, will merge thing (PP with every kind of each 15 mer peptides (5 μ g/ml), peptide from body DC (20,000/ hole); Every kind of peptide of 5 μ g/) or minimum epi-position (the ME) (D466:CFP10 that determines from the T cell clone that stems from every kind of donor _2-11D480:CFP10 _3-11D481:CFP10 _75-835 μ g/ml) pulse, and at the CD8 of 250,000 magnetic beads purifying ⁺The T cell is tested.After cultivating 18 hours altogether, assess the release of IFN-γ by ELISPOT.The mean value of two parts of replicate determinations of each some expression.

Fig. 6 is one group of figure that has summarized minimum epitope mapping data.In order to determine minimum epi-position, will be from the peptide pulse of body LCL (20,000/ hole) with indicating concentration, and with the common cultivation of T-cell (1000 cells/well).After cultivating 18 hours altogether, assess IFN-γ by ELISPOT.The mean value of two parts of replicate determinations of each some expression.

Fig. 7 is the line chart that has shown D504 clone's minimum epitope mapping.In order to determine minimum epi-position, will cultivate altogether with the T-cell clone (1000 cells/well) and the peptide of the concentration of indicating from body LCL (20,000/ hole).After cultivating 18 hours altogether, assess IFN-γ by ELISPOT.The mean value of two parts of replicate determinations of each some expression.

Fig. 8 has shown that suffering from outer (EP) TB of lung compares the figure of Mtb specific C D8+T cell response with Uganda children that suffer from intrathoracic (IT) TB.Use interferon (IFN)-γ specificity ELISPOT and use ESAT-6 and the CFP-10 peptide is originated as antigen, in the age is 10 years old or following Uganda children, measure Mtb specific C D8+T cell response.Children suffer from EP (n=35) or IT TB (n=43).TB colony mainly constitutes (30/35[86%]) by scrofula (scofula).The spot that the result is shown as the peripheral blood lymphocytes (PBMC) of per 250,000 poor CD4/CD56 forms unit (SFU).Carry out two parts of replicate determinations, positive response is defined as being higher than replying of nutrient culture media 2 standard deviations of contrast.

Fig. 9 is a process flow diagram, described enlist, the experimenter gets rid of and execution and the analysis of ELISPOT.* be meant the age group that in HE analyzes CP TB, comprises.

Figure 10 has shown up to the CD8 of the exposure children contactee all ages of 15 years old health and the figure of the comparison that CD4ELISPOT replys.Shown that the spot that is higher than background in per 250,000 T cells forms unit.Initially enlisting digital display is shown among Fig. 9 a.Carried out Cochran Armitage trend test: for CD8 ELISPOT, p=0.055; For PBMCELISPOT, p=0.2.

Figure 11 a has shown the figure of the ratio that positive ELISPOT analyzes in Uganda children by clinical research component layers≤10 years old.The CD8 and the PBMC t cell response of TB (C-TB) subgroup of having described HE and having made a definite diagnosis.Analyze for CD8, the children that suffer from C-TB obviously more may have positive (p=0.001) [the HE children are 20% (CI 0.09-0.34), than C-TB children's 58% (CI 0.37-0.77)] of analyzing.When comparing CP-TB and HE, also noticed this discovery.Similarly, analyze the positive ratio of analyzing higher (p=0.02) in the clinical subgroup of C-TB [the positive HE children that analyze are 37% (CI0.24-0.50), than C-TB children's 65% (CI 0.42-0.83)] for PBMC.Different with the CD8 analysis is that when CP-TB compared with HE, positive ratio and HE colony did not have notable difference.

The figure of Figure 11 b ratio that positive ELISPOT analyzes in by clinical research group and Uganda children by age stratification≤10 years old.Analyze for the CD8 among≤5 years old children, the children that suffer from the TB that makes a definite diagnosis compare with HE more may have positive CD8ELISPOT (p=0.009) [the HE children are 12% (CI 0.03-0.31), than C-TB children's 47% (CI 0.24-0.71)].Similarly, when CP-TB colony compared with HE, CP-TB had obviously higher positive CD8 and analyzes ratio.Compare by in≤5 years old children PBMC being analyzed, positively analyze uncorrelated with the clinical research group [the HE children are 37% (CI 0.21-0.55), and C-TB is 56% (CI 0.30-0.78)], no matter using C-TB still is that CP-TB compares and is not always the case.For＞5 years old children, quantity was few, did not therefore compare statistics.But two kinds of analyses all identify children's [CD8 for＞5 years old children analyzes, and having the positive HE that analyzes is 30% (CI 0.11-0.54), than C-TB children's 86% (CI 0.0.42-0.99)] of a high proportion of C-TP of suffering from.By analysis compared to CD4 among the children at＞5 years old, having the positive HE that analyzes is 36% (CI 0.17-0.59), and the C-TB children are 100% (CI 0.47-1.0).

Figure 12 a-12d is a set of diagrams, wherein CD8ELISPOT result for≤5 years old children (12A) and＞spot that 5 years old children (12C) are expressed as being higher than predetermined cutoff value forms unit (SFU).The children's of≤5 years old and＞5 years old PBMC ELISPOT result be presented at (12B) and (12D) in.Analyze by cd8 t cell ELISPOT, suffer from CP-TB or C-TB≤Uganda children of 5 years old have remarkable and strong replying, and healthy exposure children do not show this replying (12A).By relatively, existing in the HE contactee can be by replying that PBMCELISPOT measures, and this replying on value do not have difference with the children (12B) that suffer from CP-TB or C-TB.When using the predetermined cutoff value classification analysis, suffer from that make a definite diagnosis or possible TB≤5 years old children more may have positive CD8ELISPOT (p=0.01), yet not have the classification relevance with PBMC ELISPOT and clinical subgroup.For＞5 years old children,, do not carry out the comparison of value and statistic of classification, but shown SFU for illustration purposes because it is few to be used for the C-TB group children quantity of CD8 (n=7) and PBMC (n=5).For＞5 years old group, HE and CP-TB group was compared, on the SFU value or by classification analysis do not find differences (12C and 12D).The statistical analysis of value uses the bilateral rank test of wilxocon; For classification analysis, carried out chi-square analysis.

Sequence table

Nucleic acid of listing in appended sequence table and amino acid sequence, use are abridged as the standard alphabet of defined nucleotide base among the 37C.F.R.1.822 and amino acid whose trigram is encoded shows.Each nucleotide sequence has only shown a chain, still should be appreciated that to have comprised complementary strand when mentioning shown chain.In appended sequence table:

SEQ ID NO:1-12 is the Mtb amino acid sequence of polypeptide.

SEQ ID NO:13-14 is the amino acid of Mtb peptide.

SEQ ID NO:15-25 is the nucleotide sequence of the polynucleotide of coding Mtb polypeptide.

SEQ ID NO:26-38 is the amino acid sequence of specific Mtb epi-position.

Specific CFP10 that SEQ ID NO:39-83 is to use and ESAT6Mtb amino acid sequence of polypeptide.

SEQ ID NO:84 is the amino acid sequence of exemplary adapter.

Describe in detail

The method that is used for detecting at object m tuberculosis infection is disclosed.To as if children or suffer from the object of LTBI.Method comprises detection T cell, the particularly existence of the CD8+T cell of specific recognition Much's bacillus (Mtb) polypeptide.Method comprises and is used for the reactive CD8 of detection of biological sample ⁺The analyzed in vitro method of the existence of T cell, and comprise the body inner analysis method that detects delayed allergy.These methods are used for detecting tuberculosis children, comprise pulmonary tuberculosis and extrapulmonary tuberculosis.These methods also are used for detecting extrapulmonary tuberculosis the adult who suffers from the sick infection of latent tuberculosis.

Term

Unless otherwise noted, otherwise technical term use according to conventional usage." gene V " (Genes V) that the definition of the common term of molecular biology is found in the Beniamin Lewin that Oxford University Press (Oxford University Press) 1994 publishes (ISBN0-19-854287-9); The chief editors' such as Kendrew of Blackwell Science Ltd. company publication in 1994 " molecular biology encyclopedia " (The Encyclopedia of Molecular Biology) (ISBN 0-632-02182-9) and VCH Publishers are in the Robert A.Meyers chief editor that Inc. company publishes nineteen ninety-five " molecular biology and biotechnology: comprehensive desktop reference " (Molecular Biology and Biotechnology:a Comprehensive Desk Reference) (ISBN 1-56081-569-8).

For the ease of checking various embodiment of the present disclosure, provide the explanation of following concrete term:

Adjuvant: be used to increase antigenic medium.Adjuvant comprises the suspension of the mineral matter of adsorption antigen (alum, aluminium hydroxide or phosphate) thereon; Or water-in-oil emulsion, wherein antigenic solution is emulsified in the mineral oil (Fu Shi (Freund) Freund), includes the mycobacterium (Freund's complete adjuvant) that kills sometimes with further increase antigenicity (suppress the degraded of antigen and/or cause that macrophage flows into).Immunostimulatory oligonucleotide (oligonucleotides that for example comprises the CpG motif) also can be used as adjuvant (for example referring to U.S. Patent No. 6,194,388, U.S. Patent No. 6,207,646, U.S. Patent No. 6,214,806, U.S. Patent No. 6,218, and 371, U.S. Patent No. 6,239,116, U.S. Patent No. 6,339,068, U.S. Patent No. 6,406, and 705 and U.S. Patent No. 6,429,199).Adjuvant comprises biomolecule (" biological adjuvant "), for example costimulatory molecules.Exemplary adjuvant comprises IL-2, RANTES, GM-CSF, TNF-α, IFN-γ, G-CSF, LFA-3, CD72, B7-1, B7-2, OX-40L and 41BBL.

Amplification: be meant that for nucleic acid molecules (for example DNA or RNA molecule) operation technique increases the copy number of sample amplifying nucleic acid molecule.The example of amplification is a polymerase chain reaction, wherein contacts under the biological sample that will collect from object and the Oligonucleolide primers condition to the nucleic acid-templated hybridization permission primer and sample.Primer extends under the condition of being fit to, dissociates and then anneal, extends and dissociate with template, with the copy number of amplification of nucleic acid.Amplified production can and/or use standard technique to carry out nucleic acid sequencing by electrophoresis, restriction enzyme cutting pattern, oligonucleotide hybridization or connection and characterize.Other examples of amplification comprise for example in U.S. Patent No. 5,744, disclosed strand displacement amplification in 311, in U.S. Patent No. 6,033, in 881 disclosed nothing transcribe isothermal duplication, in WO 90/01069 disclosed reparation chain reaction amplification, disclosed ligase chain reaction amplification in EP-A-320308, in U.S. Patent No. 5,427, disclosed gap filling ligase chain reaction amplification and in U.S. Patent No. 6 in 930, disclosed NASBA in 025,134 ^TMRNA does not have transcription amplification.

Antigen: can comprise the composition of injecting or absorbing in the animal at compound, composition or the material of generation of animal moderate stimulation antibody or t cell response.The product of antigen and specificity humoral or cellular immunity, comprise the product reaction of inducing by heterogenous immunogen.Term " antigen " comprises the antigenic epitopes that all are relevant." epi-position " or " antigenic determinant " is meant on the antigen that B and/or T cell are to its site of replying.In one embodiment, when epi-position presents when combining with the MHC molecule, the T cell is replied epi-position.Epi-position can be formed by adjacent amino acids or three grades of folding and juxtaposed non-conterminous amino acid by protein.The epi-position that is formed by adjacent amino acid is typically kept after being exposed to the sex change solvent, and typically loses after with the sex change solvent processing by three grades of epi-positions that are folded to form.Epi-position typically comprises at least 3, more generally at least 5, about 9 or about 8-10 amino acid that is in unique steric configuration.The method of determining the steric configuration of epi-position comprises for example x-radiocrystallography and two dimensional NMR.

Antigen can be tissure specific antigen or disease specific antigen.These terms are not exclusiveness, because tissure specific antigen also can be a disease specific antigen.Tissure specific antigen is expressed in a limited number of tissues, for example single organization.Tissure specific antigen can be by more than one tissue expressions, such as but not limited to the antigen of in more than one germinal tissue, for example expressing in prostate and uterine tissue.The expression and the lysis of disease specific antigen are synchronous.The concrete limiting examples of disease specific antigen is that it is expressed relevant with tuberculosis or indicates antigen lungy.Disease specific antigen can be by the antigen of T cell or B cell recognition.The Mtb specific antigen has specificity to Mtb.

Antibody: the immunocompetence part of immunoglobulin molecules and immunoglobulin molecules, promptly contain the molecule that combines the antigen binding site of (generation immune response) with antigen, for example Mtb polypeptid specificity.

Naturally occurring antibody (for example IgG, IgM, IgD) comprises 4 polypeptied chains, by interconnected two heavy chains of disulfide bond (H) and two light chains (L).But, show that the antigen combined function of antibody can be carried out by the fragment of naturally occurring antibody.Therefore, these Fabs also planned to indicate in term " antibody ".Concrete, the non-limiting instance of the binding fragment that term antibody is contained comprise that (i) is by V _L, V _H, C _LAnd C _H1The Fab fragment that domain constitutes; (ii) by V _HAnd C _H1The F that domain constitutes _dFragment; (iii) by the V of the single armed of antibody _LAnd V _HThe Fv fragment that domain constitutes; (iv) by V _HThe dAb fragment (Ward etc., Nature 341:544-546,1989) that domain constitutes; (the v) complementarity-determining region of Fen Liing (CDR); And (vi) F (ab ') ₂Fragment is included in the divalence fragment of hinge area by two continuous Fab fragments of disulfide bond.

Immunoglobulin (Ig) and some variant thereof are known, and arranged many preparation in the recombinant cell culture (referring to for example U.S. Patent No. 4,745,055; U.S. Patent No. 4,444,487; WO 88/03565; EP 256,654; EP 120,694; EP 125,023; Faoulkner etc., Nature 298:286,1982; Morrison, J.Immunol.123:793,1979; Morrison etc., Ann Rev.Immunol 2:239,1984).

Animal: the many cells vertebra biology of living, this classification comprises for example mammal and birds.Term mammal comprise human and non-human mammal the two.Similarly, term " object " comprises human and animal doctor's object." children " are that the age is less than about 18 years old human subjects.In certain embodiments, " child " is that the age about 1 is to about 5 years old human subjects." juvenile " is that the age about 6 is to about 12 years old human subjects." baby " is the age less than 1 years old human subjects." teenager " is that the age about 13 is to about 18 years old human subjects." preadolescence object " do not experience puberty as yet, is that the age is less than about 11 years old human subjects in some instances.

Antigen presenting cell (APC): can be to T presented by cells antigen so that the cell of activated T cell.Dendritic cell is that the major antigen that participates in primary immune response is delivery cell (APC).Their major function be obtain in the tissue antigen, to lymphoid organ migration and antigen-presenting, so that activated T cell.

When receiving suitable maturation instruction, the dendritic cell received signal promotes the startup and the development of immune response to experience form and physiological change fast.Comprising raising the molecule that participates in antigen presentation; Generation is for generating Th1 crucial pro-inflammatory cytokine replying, comprising IL-12; And secretion helps to drive the chemotactic factor (CF) of inmature Th cell differentiation, amplification and migration on every side.In general, the molecule of these rises has promoted dendritic cell to coordinate finally to provide the activation of other peripheral lymphoid cells of protection and the ability of effector function for the host.

CDNA (complementary DNA): the DNA of one section regulating and controlling sequence that lacks inner noncoding region section (introne) and determine to transcribe.CDNA synthesizes by the reverse transcription from the mRNA of cell extraction in the laboratory.

CD4: the differentiation bunch factor 4, the T cell surface protein of a kind of mediation and II class MHC interaction of molecules.At CD4 between the HIV infection period also as the major receptors site of HIV on the T cell.The cell of expression CD4 is helper T lymphocyte normally.

CD8: the differentiation bunch factor 8, the T cell surface protein of a kind of mediation and I class MHC interaction of molecules.The cell of expression CD8 is cytotoxic T cell normally." immunity that CD8+T is cell-mediated " is the immune response of carrying out by to CD8+T presented by cells antigen.

CDNA (complementary DNA): the DNA of one section regulating and controlling sequence that lacks inner noncoding region section (introne) and determine to transcribe.CDNA synthesizes by the reverse transcription from the mRNA of cell extraction in the laboratory.

Conservative variant: " guarding " aminoacid replacement is the active or antigenic aminoacid replacement that does not influence or reduce the mycobacterium polypeptide basically.Conservative concrete, the non-limiting instance that replaces comprises following example:

The term conservative variations also comprises uses the amino acid that replaces to replace unsubstituted parent's amino acid, condition be the antibody that produces at the polypeptide that replaces also with unsubstituted polypeptide generation immune response, or the immune response that produces at the polypeptide that replaces is to for example the immune response of antigen of mycobacterium is similar at not replacing polypeptide.Therefore, in one embodiment, non-conservative replacement is to reduce active or antigenic replacement.

Basically by ... constitute/by ... constitute: for polypeptide, do not comprise any additional amino acid residue, constitute by specified aminoacid sequence basically if be meant polypeptide.But polypeptide can comprise additional non-peptide class component, for example label (for example fluorescence, radioactivity or solids label), carbohydrate or lipid.The polypeptide that is made of specified aminoacid sequence does not comprise any additional amino acid residue, does not contain additional non-peptide class component for example lipid, carbohydrate or label yet.

Contact: there is the process of carrying out incubation under the situation of another kind of reagent in a kind of reagent.Therefore, when cell contacted with reagent, cell and reagent incubation time enough section made reagent and cell interaction.

Costimulatory molecules: although TCR has sent a signal with engaging to the T cell of peptide-MHC, this independent signal may be not enough to activating T cell.Costimulatory molecules is to send to make the T cell be activated the molecule of second required signal when combining with its part.The costimulatory molecules of knowing of behaving most on the T cell is CD28, and it combines with B7-1 (being also referred to as CD80) or B7-2 (being also referred to as CD86).Other costimulatory molecules is B7-3.Also, the T cell activation comprises adhesion molecule in the cell (ICAM-1 and ICAM-2), leucocyte function-associated antigen (LFA-1, LFA-2 and LFA-3) for providing the accessory molecule of secondary signal.Integral protein and TNF (TNF) superfamily member also can be used as costimulatory molecules.

Cell factor: influence for example albumen of lymphocytic behavior of other cells by what cell was made.In one embodiment, cell factor is a chemotactic factor (CF), and a class influences the molecule of the transportation of cell.The concrete limiting examples of cell factor comprises interleukins (IL-2, IL-4, IL-6, IL-10, IL-21 etc.) and interferon (IFN)-γ.

The degeneracy variant: the polynucleotide of the epi-position of coding Mtb polypeptide comprise the sequence of degeneracy owing to the reason of genetic code.Have 20 kinds of natural amino acids, their major parts are specified by more than one codons.Therefore, as long as the nucleotide sequence of all degeneracys constant by described nucleotide sequence coded Mtb amino acid sequence of polypeptide, is included in the disclosure.

Dendritic cell (DC): dendritic cell is that the major antigen that participates in primary immune response is delivery cell (APC).Dendritic cell comprises thick liquid cell sample dendritic cell and marrow sample dendritic cell.Their major function be obtain in the tissue antigen, to lymphoid organ migration and antigen-presenting with activating T cell.Jejune dendritic cell produces in marrow, and resides in periphery as immature cell.

Diagnosis: identify that pathological condition is such as but not limited to existence lungy or character.Diagnostic method is different aspect its sensitivity and specificity." sensitivity " of diagnostic analysis is the percent (percent of true positives) of the positive diseased individuals of test." specificity " of diagnostic analysis is 1 to deduct false positive rate, and wherein false positive rate is defined as testing the ratio of the individuality of the positive but disease that do not take a disease.Although concrete diagnostic method can not provide the decisive diagnosis of illness, if method provides the positive that helps to diagnose to indicate just enough." prognosis " is meant for example probability of tuberculosis development (for example seriousness) of prediction pathological condition.

Show: with peptide: antigenic compound or peptide are positioned the process on the cell outer surface, wherein peptide: molecule or soluble factor that antigenic compound or peptide can show by second cell, by second cell are approaching.Peptide or peptide: antigenic compound, on being present in cell outer surface and the molecule that can show or soluble factor by second cell, by second cell near the time, by cell " demonstration ".

Epi-position: antigenic determinant.They are to have antigenicity, promptly cause particular chemical group or peptide sequence on the molecule of specific immune response.Antibody specificity is in conjunction with the polypeptide specific antigen epi-position on the mycobacterium polypeptide for example.

Expression control sequenc: the nucleotide sequence of the expression of regulation and control and its heterologous nucleic acid sequence that can be operatively connected.When transcribing of nucleotide sequence and the translation under suitable situation are controlled and regulated to expression control sequenc, expression control sequenc and described nucleotide sequence can be operatively connected.Therefore, expression control sequenc can comprise initiation codon (being ATG) before the promoter, enhancer, transcription terminator, protein coding gene, introne splicing signal, keep the proper reading frame of described gene to allow correct translation and the terminator codon of mRNA.Term " control sequence " is planned bottom line and is comprised the element that its existence can influence expression, and also can comprise it and have other favourable elements, for example targeting sequencing and fusion partner sequence.Expression control sequenc can comprise promoter.

Promoter is to be enough to instruct the minmal sequence of transcribing.Also comprise and be enough to make the expression of promoter dependent gene maybe can be subjected to external signal or medicament to induce controllable promoter element at cell type specificity, tissue specificity; These elements can be arranged in 5 of gene ' or 3 ' district.Composing type and inducible promoter (referring to for example Bitter etc., Methods in Enzymology 153:516-544,1987) have been comprised.In the time of for example in being cloned into bacterial system, can use inducible promoter for example pL, plac, ptrp, the ptac (ptrp-lac hybrid promoter) etc. of bacteriophage lambda.In one embodiment, in the time of in being cloned into mammal cell line system, can use to stem from mammalian cell genome (for example metallothionein promoter) or stem from mammalian virus (retroviruse long terminal repeat for example; Gland virus stage starting; Vaccinia virus 7.5K promoter) promoter.Also can use the promoter that produces by recombinant DNA or synthetic technology that transcribing of nucleotide sequence is provided.In one embodiment, promoter is a cytomegalovirus promoter.

Classification: sample experience is separated the component of sample according to physics or chemical property, such as but not limited to size, electric charge, solubleness or composition conditioned disjunction process.The example of classification process includes but not limited to for example ion-exchange chromatography of selective precipitation, organic extraction, size exclusion dialysis or chromatography.In one embodiment, level part is for example soluble extract or the organic extract of mycobacterium of biosome.

Function equivalence: produce and described identical result's sequence variation, for example sequence variation in the epitope herein.Such sequence variation can include but not limited to conservative replacement, disappearance, sudden change, frameshit and insertion.

Allos: stem from different genetic origins or species.Stem from the nucleic acid of the Mtb polypeptide of not encoding with the polypeptide of Mtb polypeptide allos.In concrete a, non-limiting instance, polypeptide comprises from 9 continuous amino acids of Mtb polypeptide or from maximum 20 continuous amino acids of Mtb polypeptide, and the allogeneic amino acid sequence comprises beta galactosidase, maltose-binding protein, albumin, hepatitis B surface antibody or immunoglobulin amino acid sequence.In general, the antibody that combines with the target protein specificity will not combine with the heterologous protein specificity.

Host cell: the cell that carrier can be bred therein and its DNA can express therein.Cell can be protokaryon or eucaryon.Cell can be mammal, human cell for example.This term also comprises the spawn of object host cell.Should be appreciated that all offsprings can be not consistent with parental cell, because may undergo mutation between replicative phase.But, when using term " host cell ", comprised such offspring.

Humam leucocyte antigen (HLA): the science of heredity title of human main histocompatibility complex (MHC).Each locus indicated by capitalization, and for example in HLA-E, and allele indicates with numeral, for example in HLA-A*0201.Three kinds of main I class mhc genes are called as HLA-A, HLA-B and HLA-C.But coding has many with gene with the chain β2Wei Qiudanbai relevant cell surface molecular of I class mhc gene.Aspect the expression of these expression of gene on Tissue distribution and cell is variable; These genes are called as IB class mhc gene.

Immune response: immune cell is B cell, natural killer cell or T cell replying stimulation for example.In one embodiment, replying specific antigen is specific (" antigentic specificity is replied ").In one embodiment, immune response is a t cell response, and for example Th1, Th2 or Th3 reply.In another embodiment, immune response is replying of suppressor T lymphocyte.

Immunogenic peptide: peptide comprises allele-specific motif or other sequences, makes described peptide combine with the MHC molecule and inducing T cell is replied, CD8 for example ⁺T cell response, or at the B cell response (for example antibody producing) of the antigen that produces immunogenic peptide.In other examples, immunogenic peptide is from CD8 ⁺The induced t cell cell factor produces.

In one embodiment, immunogenic peptide uses sequence motifs or additive method known in the art for example neural network or the evaluation of polynomial expression determination method.Typically, use algorithm to determine " in conjunction with the threshold value " of peptide, with select to have give they with certain compatibility combination high probability and will be the peptide of immunogenic score value.Algorithm is based on the specific amino acids of ad-hoc location to the influence of the specific amino acids antagonist combination of the influence of MHC combination, ad-hoc location or contain in the peptide of motif specific replacement to the influence of combination.In the situation of immunogenic peptide, " conserved residues " is that the frequency ratio of the specific location appearance of this residue in peptide is obviously higher by the desired frequency of stochastic distribution.In one embodiment, conserved residues is that wherein the MHC structure can provide residue with the contact point of immunogenic peptide.

Immunogenic peptide also can be by measuring they and the combining and stimulate CD8 when existing under the situation of MHC albumen by them of specific MHC albumen ⁺The ability of T cell is identified.In an example, immunogenicity " Mtb peptide " is a series of adjacent amino acids residues from Mtb albumen, and general length is between 9 to 20 amino acid, and for example length is about 8 to 11 residues.Disclosed in this article specific immunogenic polypeptide length is 9 or 10 amino acid residues, or length is maximum 12 amino acid.

In general, immunogenicity Mtb polypeptide is used in induce immune response in the object, for example B cell response or t cell response.In an example, immunogenicity Mtb polypeptide is when when the main histocompatibility complex of I class molecule combines, activation CD8 ⁺The T cell is for example at the cytotoxic T lymphocyte (CTLs) of Mtb.Use synthetic inducing peptide CTL and CTL cytotoxicity analysis method known in the art, referring to United States Patent (USP) 5,662,907, it draws at this and is reference.In an example, immunogenic peptide comprises allele-specific motif or other sequences, makes peptide and to induce CD8 at the antigen that produces immunogenic peptide in conjunction with the MHC molecule ⁺Reply.The CD8 of specific recognition Mtb polypeptide ⁺The T cell is made response, activation, propagation and/or secrete cytokines to specific polypeptide rather than other uncorrelated polypeptide.

Immunogenic composition: comprise the composition of the nucleic acid of immunogenicity Mtb polypeptide or coding immunogenicity Mtb polypeptide, it induces the measurable t cell response at Mtb, for example CD8 ⁺T cell response, or induce measurable B cell response (for example specificity is in conjunction with the production of the antibody of Mtb polypeptide).For external application, immunogenic composition can by the nucleic acid that separates, comprise the carrier of nucleic acid/or immunogenic peptide constitute.The planted agent uses for body, and immunogenic composition typically comprises nucleic acid, comprises the carrier and/or the immunogenic polypeptide of nucleic acid in pharmaceutically suitable carrier and/or other reagent.Immunogenic composition can be chosen the nucleic acid that comprises adjuvant, costimulatory molecules or coding costimulatory molecules wantonly.Can easily test Mtb polypeptide or nucleic acid encoding and induce the ability of CD8+T cell response.

Suppress or the treatment disease: inhibition disease for example tuberculosis is meant the development in an all-round way that suppresses disease.In several examples, the inhibition disease is meant and alleviates symptom lungy." treatment " be meant the S or S that improves disease or with the therapeutic intervention of the pathological condition of disease association, described disease is tuberculosis for example.

Gamma interferon: IFN-γ is a dimer protein, and its subunit has 146 amino acid.By glycosylation, pI is 8.3-8.5 to albumen two site.IFN-γ is synthetic as 166 amino acid whose precursor proteins, and it comprises 23 amino acid whose secretory signal sequences.Described 20 and the biological activity protein of two kinds of molecular forms of 25kDa.They the two all 25 glycosylations.The 25kDa form is also 97 glycosylations.Natural IFN-γ is because variable glycosylation pattern in observed difference aspect molecular mass and the electric charge.Observed 40-60kDa form is IFN-γ under non-sex change condition the dimer and the tetramer.Human gene has the length of about 6kb.It comprises four extrons, and is plotted on chromosome 12q24.1.

IFN-γ can detect by sensitive immunoassay, for example allows to detect the ELSA test of each cell that produces IFN-γ.A spot of IFN-γ can by measure albumen that IFN induces for example Mx albumen come indirect detection.IP-10 is synthetic induces the concentration that also can be used for measuring IFN-γ.In addition, bioanalysis can be used for detecting IFN-γ, for example utilizes in the 2D9 cell and induces indoleamine 2, the analysis of 3-dioxygenase activity.The generation of IFN-γ can be used for assessing the T cell activation, and for example the T cell is activated by the antigen of mycobacterium that HLA-E presents.

Separate: " separation " nucleic acid with the natural biological cell that has nucleic acid in other nucleotide sequences, be other chromosomes and exchromosomal DNA and RNA separates basically or purifying comes out.Therefore, term " separation " comprises the nucleic acid by the nucleic acid purification method purifying of standard.This term also comprises the nucleic acid by recombinant expressed nucleic acid for preparing and chemosynthesis in host cell.

Label: but directly or indirectly engage detection compound or composition with another molecule with the detection that promotes this molecule.Concrete, the non-limiting instance of label comprises that fluorescence labels, enzyme connect and radioactive isotope.

Joint sequence: joint sequence is the amino acid sequence in covalently bound two polypeptide structure territories.Joint sequence can be included between the Mtb epi-position disclosed herein, for the polypeptide structure territory that is connected provides rotary freedom, and promotes folding and the presenting to MHC of the domain that is fit to thus.For example, in the recombinant polypeptide that comprises two Mtb domains, joint sequence can be provided between them, for example comprises the polypeptide of Mtb polypeptide-joint-Mtb polypeptide.The general length of joint sequence is between 2 to 25 amino acid, it is being well-known in the art, and include but not limited to by Chaudhary etc. at Nature 339:394-397 glycocoll (4)-serine sept (GGGGS (SEQ ID NO:84) x3) of describing in 1989.

Lymphocyte: a class leucocyte that participates in the immune defense of health.The lymphocyte that two kinds of main types are arranged: B cell and T cell.

Mammal: this term comprises the mankind and non-human mammal.Similarly, term " patient " or " object " comprise human and animal doctor's object.

Mycobacterium: bacterium living beings generic in the aerobic cell.Behind infection host, these biosome survivals are in the interior body structure of monocyte and macrophage.Human mycobacterium disease comprises tuberculosis ((M.tuberculosis) causes by Much's bacillus), leprosy ((M.leprae) causes by the leprosy mycobacterium), Bairnsdale canker ((M.ulcerans) causes by mycobacterium buruli) and can be by Mycobacterium marinum (M.marinum), mycobacterium kansasii (M.kansasii), scrofula mycobacterium (M.scrofulaceum), Suhl adds branch bacillus (M.szulgai), mycobacterium xenopi (M.xenopi), mycobacterium fortutitum (M.fortuitum), mycobacterium haemophilum (M.haemophilum), other infection that mycobacterium chelonei (M.chelonei) and Mycobacterium intracellulare (M.intracellulare) cause.The mycobacterium strain (for example mycobacterium avium (M.avium)) that was considered to non-pathogenic has in the past now also known it is immunosuppressant AIDS patient's main killer.

Mainly replying of mycobacterium comprised and cell-mediated super quick (DTH) reaction of T cell and macrophage that it kills and isolate (or comprising) in (granuloma formation) and plays a significant role in the cell of biosome.Main t cell response relates to the CD4+ lymphocyte of identification mycobacterium heat shock protein and immunodominance antigen.

Can be operatively connected: have functionally when related when first nucleotide sequence is placed in second nucleotide sequence, first nucleotide sequence promptly can be operatively connected with second nucleotide sequence.For example, if promoter is carried out transcribing or expressing of coded sequence, then promoter and coded sequence can be operatively connected.In general, the dna sequence dna that can be operatively connected is adjacent, and connects at needs under the situation of two protein-coding regions, and open reading frame is alignd.

ORF (open reading frame): the nucleotide triplet (codon) of a series of coded amino acids without any terminator codon.These sequences can be translated into polypeptide usually.

Peptide is modified: the mycobacterium polypeptide comprises the synthetic embodiment of peptide described herein.In addition, in method described herein, can utilize analog (non-peptide organic molecule), derivant (from disclosed peptide sequence, the peptide molecule of the chemistry functional of acquisition) and the variant (analog) of these albumen.Each polypeptide of the present invention is made of amino acid sequence, and described amino acid can be naturally occurring or the L-and/or the D-amino acid of non-natural existence.

Peptide can be modified by various chemical technologies, has the active and optional derivant with other required character substantially the same with the unmodified peptide with generation.For example, the hydroxy-acid group of albumen no matter be on carboxyl terminal or the side chain, can be provided as the form of pharmaceutically acceptable cationic salt, or esterification forms C ₁-C ₁₆Ester, or transform accepted way of doing sth NR ₁R ₂Acid amides, R wherein ₁And R ₂Be H or C independently of one another ₁-C ₁₆Alkyl, or be combined to form heterocycle for example 5 Yuans or 6 Yuans rings.The amino group of peptide no matter be on amino terminal or the side chain, can adopt the form of pharmaceutically acceptable acid addition salts, and for example HCl, HBr, acetate, benzoic acid, toluenesulfonic acid, maleic acid, tartrate and other organic acid salt perhaps can be modified into C ₁-C ₁₆Alkyl or dialkyl amido or further change into acid amides.

The hydroxyl of peptide side chain can use recognized techniques to change into C ₁-C ₁₆Alkoxy or C ₁-C ₁₆Ester.The phenyl ring of peptide side chain or phenol ring can be with one or more halogen atoms for example fluorine, chlorine, bromine or iodines or use C ₁-C ₁₆Alkyl, C ₁-C ₁₆The acid amides of alkoxy, carboxylic acid and ester thereof or these carboxylic acids replaces.The methylene of peptide side chain can prolong into the C of homology ₂-C ₄Alkylidene.Mercaptan can with in numerous generally acknowledged blocking groups any, for example acetamide group protects.The professional in present technique field also will know the method that is used for ring texture is imported to peptide of the present invention, to select and to provide the configuration to structure to restrict, produce the stability that increases.

Be susceptible to the embodiment of peptide simulation and organic simulation, make the three-dimensional arrangement simulating peptide skeleton and the three-dimensional arrangement of forming amino acid side chain of the chemical composition of these peptides and organic analogies thus, cause these peptides of mycobacterium polypeptide and the ability that organic analogies have the generation immune response that can measure or increase.Use for computer simulation, pharmacophore is that the Utopian, three-dimensional of bioactive structural requirement limited.Can use existing computer simulation software (ancillary drug that uses a computer design or CADD), peptide and organic analogies are designed to agree with each pharmacophore.For the description of the technology of in CADD, using, referring at Klegerman ﹠amp; " medicine biotechnology " (Pharmaceutical Biotechnology) of Groves chief editor in 1993, Interpharm Press:Buffalo Grove, " pharmacology principle " (Principles of Pharmacology) the 102nd chapter that the Walters of 165-174 page or leaf " computer assisted medicine simulation " (Computer-Assisted Modeling of Drugs) and Munson nineteen ninety-five edit among the IL.The analogies that use such technology preparation have also been comprised.

Medicament or medicine: chemical compound or the composition that when suitably delivering medicine to object, can induce required treatment or prophylactic effect.

Pharmaceutically suitable carrier: the pharmaceutically suitable carrier that can be used for polypeptide described herein and nucleic acid is conventional." the Remington materia medica " of E.W.Martin (Remington ' s Pharmaceutical Sciences), Mack Publishing Co., Easton, PA the 15th edition (1975) has described composition and preparation that the medicine that is suitable for fusion disclosed herein is sent.

In general, the character of carrier depends on employed concrete administering mode.For example, parenteral administration comprises injectable fluid usually, it comprise the pharmaceutically acceptable and acceptable fluid of physiology for example water, physiological saline, balanced salt solution, G/W, glycerine etc. as medium.For solid composite (for example pulvis, pill, tablet or capsule form), conventional avirulence solid carrier can comprise for example pharmaceutical grade mannitol, lactose, starch or dolomol.Except the bio-neutral carrier, treat that the pharmaceutical composition of administration can comprise a small amount of avirulence auxiliary substance, for example wetting or emulsifying agent, antiseptic and pH buffering agent etc., for example sodium acetate or sorbitan monolaurate.

Polynucleotide: the linear kernel nucleotide sequence, it comprises that length is longer than the sequence of 100 nucleotide bases.

Polypeptide: any amino acid chain, irrelevant length or posttranslational modification (for example glycosylation or phosphorylation)." peptide " is that length is less than 100 amino acid whose amino acid chains.In one embodiment, " peptide " is the part of polypeptide, and for example length surpasses about 10,20,30,40,50 or 100 continuous amino acids of 100 amino acid whose polypeptide.

The part of nucleotide sequence: at least 10,20,30 or 40 continuous nucleotide of the sequence of correlated series, for example coding for antigens.In some cases, it will be favourable using the part that is made of 50 or above nucleotide.For example, when describing the part (for example antigenic epitopes) of antigen, it may be favourable removing the part that correlated series comprises at least 10,20,30,40 or 50 nucleotide according to length.

Probe and primer: nucleic acid probe and primer can preparations easily on the basis of nucleic acid provided by the invention.Probe comprises the nucleic acid with the separation of detectable or reporter molecules.Typical label comprises radioactive isotope, part, chemiluminescence agent and enzyme.Be used for the method for mark and select to be suitable for the guilding principle of the label of various purposes, in (1987) such as for example Sambrook etc. (1989) and Ausubel, discuss.

Primer is short nucleic acid, and being preferably length is 15 nucleotide or above DNA oligonucleotides.Primer can to form crossbred between primer and target dna strand, be extended along target dna strand by archaeal dna polymerase then by nucleic acid hybridization and complementary target dna strand annealing.By for example polymerase chain reaction (PCR) or other nucleic acid amplification methods known in the art, can use primer to amplifying nucleic acid sequence.

Be used to prepare and use the method for probe and primer, be described in " molecular cloning experiment guide " second edition (Molecular Cloning:A Laboratory Manual of chief editor such as Sambrook for example, 2nd ed)., vol.1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, " molecular biology modernism " (Current Protocols in Molecular Biology) with chief editor such as Ausubel, Greene Publishing and Wiley-Interscience, New York is in 1987 (regular updates).The PCR primer for example is intended for use the computer program of this purpose to deriving out from known array by use, for example Primer (0.5 edition,

1991, Whitehead Institute for Biomedical Research, Cambridge, MA).

The prevention or the treatment disease: " prevention " disease be meant for example known be in by the people under the risk of Much's bacillus or leprosy mycobacterial infections in the inhibition disease comprehensive generation.Example with people of known physique is and is suffered from the contubernal people of people lungy, hygiene care professional by diagnosis, is exposed to the people of Much's bacillus in the family or." the preventive activities sexuality is dyed " is meant and prevents that latent infection is transformed into tuberculosis.

" treatment " is meant the therapeutic intervention that improves its S or S in disease or pathological condition after for example tuberculosis has begun to take place.

Promoter: promoter is to instruct a series of nucleic acid control sequences of transcribed nucleic acid.Promoter comprises the essential nucleotide sequence near the initiation site of transcribing, and for example is the TATA element under the situation of II type polymerase promoter.Also optional enhancer or the repressor element at a distance that comprise of promoter, it can be positioned at apart from transcription initiation site and reach the right position of several kilobase.Promoter can be composing type or inducible promoter.Concrete, the non-limiting instance of promoter are HCMV IE promoters.

Purifying: the term purifying do not need absolute purity; On the contrary, it is planned as relative terms.Therefore, for example, the antigen preparation thing of purifying be wherein antigen than albumen purer antigen preparation thing in its intracellular primal environment.Antigen preparation thing typical case carries out purifying, makes antigen account at least 50% of prepared product total protein content.But, for some is used, may need more highly purified prepared product.For example, for such application, can use antigen wherein to account at least 75% or at least 90% prepared product of total protein content.In some instances, the antigen of purifying accounts at least 90%, at least 95%, at least 98% or at least 99% of total protein content.

Reorganization: the nucleic acid of reorganization or polypeptide are nucleic acid or the polypeptide with sequence that the sequence that non-natural exists or the artificial combination with the sequence section that separates by two or more scripts make.This artificial combination is often by chemosynthesis or more common manually-operated by the nucleic acid segment of separating, for example realize by genetic engineering technology.

Sequence homogeneity: the similarity between the amino acid sequence is represented according to the similarity between the sequence, is also referred to as sequence homogeneity.Sequence homogeneity is often measured according to homogeneity (or similarity or homology) percentage; Percentage is high more, and two sequences are similar more.When using standard method to compare, the variant of antigen polypeptide will have high relatively sequence homogeneity degree.

The method that aligned sequences is used for comparison is being known in the art.Altschul etc. (1994) have proposed the detailed consideration of sequence alignment method and homology calculating.The basic local comparison research tool (Basic Local Alignment Search Tool) of NCBI is (Altschul etc. (BLAST), 1990) can obtain from several sources, comprise (the NCBI of NCBI (National Center for Biotechnology Information), Bethesda, MD) and on the internet, be used for being used in combination with sequential analysis program blastp, blastn, blastx, tblastn and tblastx.It can be at the NCBI website visiting.How to use this program to determine that the description of sequence homogeneity can obtain on the NCBI website, default parameter also is like this.

Antigenic polypeptide is the mycobacterium variant polypeptides for example, use NCBI Blast 2.0, promptly be set to the gap blastp of default parameter, when carrying out the total length comparison, typically be characterised in that sequence homogeneity with calculating of at least 50% with the amino acid sequence of native antigen sequence.When this method of use is assessed, the albumen that has a higher similarity with reference sequence will demonstrate the percentage homogeneity of increase, for example at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 90% or at least 95% sequence homogeneity.When carrying out sequence homogeneity relatively the time to being less than whole sequence, variant typically has at least 75% sequence homogeneity in 10-20 amino acid whose short window, and depend on the similarity of they and reference sequence, may have at least 85% or at least 90% or 95% sequence homogeneity.Be used in so short window, determining that the method for sequence homogeneity is described in the NCBI website.MHC domain variant polypeptides also keeps the biologically active of natural polypeptides.For purposes of the present invention, by the variant structure territory being mixed in suitable β 1 α 1 or α 1 α 2 polypeptide, and measure the polypeptide obtain according to following detailed in vitro inhibition antigen specific T-cell proliferation or induce the ability of the expression of T SC or IL-10, assess this activity easily.

Therapeutic activity polypeptide: the medicament that causes the inducing of immune response (for example the molten cytoactive increase of the increase of immune cell population, the anti-Mtb infection symptoms that maybe can measure alleviates) that measures by clinical response, for example Mtb epi-position.The therapeutic activity molecule also can be made by nucleic acid.Example based on the therapeutic activity molecule of nucleic acid is the nucleotide sequence of coding Mtb epi-position, wherein nucleotide sequence and control element for example promoter can be operatively connected.

In one embodiment, the treatment effective dose of Mtb polypeptide is the amount that is used to produce immune response.In several examples, " treatment " is meant the therapeutic intervention that improves S or S lungy.

Treatment effective dose: be enough to stop disease progression or cause disease to go down maybe to alleviate the dosage of the symptom that causes by disease.In one embodiment, the treatment effective dose is the dosage that is enough to stop development lungy or alleviates its symptom.

Transduction and transform: when virus or carrier are transferred to nucleic acid in the cell, its " transduction " cell.When DNA being become duplicated by cytotostatic in by the genome that nucleic acid is mixed cell or by episomal replication, then cell is by the nucleic acid " conversion " in this cell of transduceing.When using in this article, term transforms and comprises the technology that all can import nucleic acid molecules this cell, comprises with the viral vectors transfection, quickens to import naked DNA with the plasmid vector conversion with by electroporation, fat transfection and particle gun.

Tuberculosis (TB) disease: a kind of disease that generally causes by m tuberculosis infection.Tuberculosis comprises lung and extrapulmonary tuberculosis.Tuberculosis is to infect the Symptomatic illness that causes by Mtb.

Pulmonary tuberculosis is the tuberculosis that is caused by Mtb.According to Center for Disease Control (Center for Disease Control), symptom generally includes cough, and can comprise cough blood or phlegm, pectoralgia, weakness, lose weight, have a fever, feel cold and night sweat.

The propagation of Much's bacillus is undertaken by the air approach in the closed region of improper ventilation.In surpassing 90% case, behind m tuberculosis infection, immune system stops the disease progression that is caused by Much's bacillus that is commonly called active tuberculosis.But not every Much's bacillus all is killed, and and then forms small hard pod membrane." primary tuberculosis " is the disease that takes place after primary infection, is common in children.Initial focus of infection is a granuloma under the little pleura, infects with granulomatous hilar lymph node.These lump together and have constituted Ghon syndrome.In nearly all case, these granulomas are cleared up, and infect not further diffusion." secondary tuberculosis " is mainly seen among the adult, as the activation again of former infection (or subinfection) again, particularly when health status goes down.Granulomatous inflammation more fully manifests and spreads.In typical case, the influenced maximum of upper lobe, and cavity formation can take place." latency " tuberculosis is can infect such as but not limited to the detected Mtb of tuberculin skin test (TST) by diagnostic assay in the individuality, wherein infects not producing symptom in this individuality." activity " tuberculosis is that Symptomatic Mtb infects in the object.

At microscopically, it is granulomatous following TB to infect the inflammation that produces, have epithelium sample macrophage and bright Han Shi (Langhans) giant cell and lymphocyte, thick liquid cell, may also have a small amount of polymorphonuclear cell, have the fibroblast of collagen, and have the characteristic caseous necrosis at the center.Inflammatory response is by IV type hypersensitivity mediation, and skin test just is based on this reaction.In some instances, tuberculosis can make up by skin test, acid-fast staining, auramine dyeing or its and diagnose.The most frequently used examination sample is a sputum, but also can carry out histological stain to tissue or other body fluid.

TB is the common complication that HIV infects.Have in the object of human immunodeficiency virus (HIV) in infection, TB infects and can easily spread and fast-developing one-tenth active disease.The concrete symptom that is infected the tuberculosis that causes by Mtb comprises long-term cough and spitting of blood.Other symptoms of TB disease comprise fatigue, lose the appetite, lose weight, have a fever and a large amount of night sweat.

Mtb infects normally, and lung infects.But, tuberculosis propagates into and can cause occurring a large amount of uncommon results with characteristic pattern outside the lung, comprises bone tuberculosis, genital tract tuberculosis, urethral tuberculosis, central nervous system (CNS) tuberculosis, stomach and intestine tuberculosis, acate pneumonic tuberculosis, scrofula and heart tuberculosis.Therefore, the MtB infection also can be that lung is outer.The outer position of the lung that infects generally includes lymph node, pleura and osteoarthrosis zone, but any organ all may be related to.The diagnosis of extrapulmonary tuberculosis often the difficulty.In general, children and immunosuppressant object infect susceptible to the outer Mtb of lung.

Lymphnoditis is the extrapulmonary tuberculosis form of normal generation.The cervical lymph node disease is the most common, all has description in groin, armpit, mesenterium, mediastinum and the breast but involve.In the U.S., tuberculosis of pleura accounts for about 5% of all tuberculosis cases.Tuberculosis of pleura often is with cough, pleura pectoralgia, fever or dyspneic acute disease.Bone and joint tuberculosis can account for nearly 35% of extrapulmonary tuberculosis case.Bone tuberculosis is usually directed to backbone most, secondly is tuberculous arthritis and the outer tuberculous osteomyelitis of vertebra in the weight-bearing joint.Tuberculosis of central nervous system comprises tubercular meningitis (modal presentation), encephalic tuberculoma and tuberculosis of spine archnoiditis.Strong inflammation after meningitis is broken in cavum subarachnoidale by tubercle under the endyma causes.The abdominal tuberculosis disease may relate to intestines and stomach, peritonaeum, lymphonodi mesenterici or genitourinary tract.Other organs (for example liver, spleen, adrenal gland) are affected in miliary tuberculosis usually.Miliary tuberculosis, tuberculous pericarditis and with tumor necrosis factor-alpha (TNF-α) tuberculosis that inhibitor is relevant, be other forms of extrapulmonary tuberculosis.Term " grain graininess " tuberculosis is meant the tuberculosis of any carrying out property, dispersivity form; This disease can be during primary be disseminated or untreated tuberculosis take place after the several years.In 10% the patient who suffers from AIDS and pulmonary tuberculosis, and in 38% the patient who suffers from AIDS and extrapulmonary tuberculosis, observed grain graininess disease.

Extrapulmonary tuberculosis for form of ownership, as initial therapy, 6 to 9 months therapeutic scheme (bimestrial isoniazid, rifampin, pyrazinamide and ethambutol carried out in recommendation, 4 to 7 months isoniazid, rifampin then), unless known or strong doubt biosome is to a line drug resistant.

Carrier: thus be directed to the nucleic acid molecules that produces transformed host cells in the host cell.Carrier can comprise permission its nucleotide sequence that duplicates, for example replication origin in host cell.Carrier can also comprise one or more selective key thing genes and other genetic elements known in the art.Carrier comprises plasmid vector, comprises being used for the plasmid of expressing at Gram-negative and gram-positive bacterium cell.Exemplary carrier comprises the carrier that is used in Escherichia coli (E.coli) and salmonella (Salmonella) expression.Carrier also comprises viral vectors, such as but not limited to retroviruse, vaccinia subgroup virus, avipoxvirus, fowlpox virus, capripox virus, pig pox virus, adenovirus, herpesviral, Alphavirus, baculoviral, this (Sindbis) of hot Derby virus, vaccinia virus and poliovirus carrier.Carrier also comprises and is used for the carrier of expressing at yeast cells.

Unless explanation is arranged in addition, all technology of using in this article and scientific terminology have with the disclosure under the identical meaning of ordinary skill institute common sense in the technical field.Indicate unless context has clearly in addition, otherwise the noun of not indicating with concrete quantity comprises its plural number.Similarly,, context indicates unless having clearly in addition, otherwise word " or " plan to comprise " with ".Should be appreciated that in addition all base sizes that provide for nucleic acid or polypeptide or amino acid size and all molecular weight or molecular mass values all are approximate values, it is provided is in order to describe.Although can be used for practice of the present disclosure or test to method similar or of equal value as herein described and material, suitable method and material be described below.Term " comprises " and means " comprising ".It is reference that all publications of mentioning in this article, patented claim, patent and other lists of references draw in full with it.Having under the situation of conflict, with this instructions, comprise that the explanation of term is as the criterion.In addition, material, method and example only are illustrative, and not plan be restrictive.

Be used to detect the method that Mtb infects: the detection of T cell

Herein disclosed is and be used for the method with the object detection mycobacterial infections of suffering from the sick infection of latent tuberculosis (LTBI) children.Children can be any children, comprise that baby, child, juvenile, age are 10 years old or following children, preadolescence children or teenager less than about 5 years old children, age.In several examples, children's age be 10 years old or below, for example the age be 7 years old or following or 5 years old or below, or the age was from 5 to 10 years old.In certain embodiments, children have with the family of TB or LTBI and contact.Family's contact is an individuality any and the common inhabitation of children.In other embodiments, to liking any doubtful object of suffering from LTBI.In an example, the object of the doubtful LTBI of suffering from is contacted with the family that has Mtb to infect, or travels to the country that the tuberculosis high incidence is arranged.

In one embodiment, method is to be used to the method that detects tuberculosis, comprise lung and/or extrapulmonary tuberculosis.Tuberculosis is to infect the Symptomatic illness that causes by Mtb.Pulmonary tuberculosis is the disease that is caused by the Mtb that causes pneumonia.Provide herein and be used for detecting the method for pulmonary tuberculosis for example children.Children can be any children, comprise that baby, child, juvenile, age are 10 years old or following children, children, preadolescence children or the teenager that the age is 5 to 10 years old less than about 5 years old children, age.Children's age can be that 6 years old or following or age were from 4 to 11 years old for 7 years old or following or age also.

Also disclose in grow up object or children and detected the method that infects outside the tuberculosis lung.Children can be any children, comprise that baby, child, juvenile, age are 10 years old or following children, children, preadolescence children or the teenager that the age is 5 to 10 years old less than about 5 years old children, age.Children's age can be that 6 years old or following or age were from 4 to 11 years old for 7 years old or following or age also.In other examples, object since hereditary illness, immunosuppressive therapy or by Immunodeficiency virus for example human immunodeficiency virus (HIV) infect and immunity is impaired.

Extrapulmonary tuberculosis can be any disease form, comprise lymphnoditis, tuberculosis of pleura, bone and joint tuberculosis, tuberculosis of central nervous system, abdominal tuberculosis, miliarytuberculosis, tuberculous pericarditis and with tumor necrosis factor-alpha (TNF-α) tuberculosis that inhibitor is relevant.Method can be used for detecting the outer tuberculous osteomyelitis of bone tuberculosis, the tuberculous arthritis in the weight-bearing joint and vertebra of vertebra.Method can be used for diagnosing tuberculosis of central nervous system, comprises tubercular meningitis (modal presentation), encephalic tuberculoma and tuberculosis of spine archnoiditis.Method also can be used for diagnosing abdominal tuberculosis disease, for example infection of intestines and stomach, peritonaeum, lymphonodi mesenterici or genitourinary tract.

In several embodiments, can be according to CD8 in the biological sample ⁺The existence of T cell detects mycobacterial infections (and/or tuberculosis), wherein T cell and Mtb polypeptid specificity reaction.In an example, with sample with one or more mycobacterium polypeptide disclosed herein, the coding polynucleotide of described one or more Mtb polypeptide and the APC incubation of expressing described one or more Mtb polypeptide or its fragment that combines with MHC.Detect CD8 ⁺The existence of T cell-specific activation or do not exist.CD8 ⁺The activation of T cell shows the existence of mycobacterial infections.In an example, by measuring cell factor, detecting CD8 such as but not limited to the expression of gamma interferon ⁺The activation of T cell.

In several embodiments, method comprises separation of C D8 ⁺The T cell.In several embodiments, the biological sample that comprises the T cell obtains from destination object.The biological sample that is fit to includes but not limited to the T cell (CD3 for example of blood sample, peripheral blood lymphocytes, sputum, saliva, cerebrospinal fluid or separation ⁺The T cell) sample, lymph node tissue, lung tissue or other tissue samples.

The CD8 of identification polypeptide in detection method ⁺The T cell is generally by in vivo to target Mtb presensitization.In several embodiments, these T cells that live through antigen are generally with 10 ⁶To 10 ³1 frequency appears in the host's who is exposed to antigen the peripheral blood in the individual peripheral blood lymphocytes (PBMC).

The T cell can separate from destination object, described destination object such as but not limited to baby, child, juvenile, age be 5 to 10 years old children, age less than 5 years old children, age less than 10 years old children, teenager, with the object of suffering from the individual common children that live of TB or LTBI, any doubtful LTBI of suffering from or doubtfully suffer from for example object of tuberculosis of tuberculosis.The T cell also can be from anyly doubtfully suffering from object that mTB outside the lung infects, comprising that children, preadolescence children and adult object separate.The T cell can separate (the Ficoll/Hypaque density gradient centrifugation by peripheral blood lymphocyte for example, or the cell sorting by fluorescence-activation) by routine techniques.In one embodiment, the T cell that uses in analytical approach is to be untreated or the form of the sample that dilutes, or the T cell of fresh separated (monocyte (MC) of the fresh separated of the use of for example directly exsomatizing or the form of peripheral blood lymphocytes (PBMC)), make them before being used for method, need not cultivate.But, also can cultivate the T cell before use, for example at one or more peptides and generally also have external source the promotion growth cell factor in the presence of.In the training period, peptide is typically presented at cell for example on the surface of APC.The pre-cultivation of T cell can cause the sensitivity of method to increase.Therefore, the T cell can be transformed into clone, for example short-term clone.

Determine that for example existence or the non-existent method of CD8 are being well-known to cell surface marker in the art.In typical case, use the labelled antibody identification of cell colony of specificity at mark.Antibody can engage other compounds, includes but not limited to enzyme, magnetic beads, colloidal state magnetic beads, haptens, fluorophore, metallic compound, radioactivity compound or medicine.The enzyme that can engage with antibody includes but not limited to alkaline phosphatase, peroxidase, urase and beta galactosidase.The fluorophore that can engage with antibody includes but not limited to fluorescein isothiocyanate, tetramethyl rhodamine isothiocyanates, phycoerythrin, allophycocyanin and texas Red.For other fluorophores that can engage with antibody, referring to Haugland, R.P. " molecular probe: fluorescence probe and research with the chemical substance handbook (Molecular Probes:Handbook of Fluorescent Probes and Research Chemicals) (1992-1994).The metallic compound that can engage with antibody includes but not limited to ferritin, collaurum and particularly colloid superparamagnetism pearl.The haptens that can engage with antibody includes but not limited to biotin, digitophyllin, azolactone and nitrophenols.The radioactivity compound that can engage or be incorporated into antibody in the antibody is known in the present technique field, and include but not limited to Tc 99m ( ⁹⁹Tc), ¹²⁵I and comprise any radioactive nuclide and include but not limited to ¹⁴C, ³H and ³⁵The amino acid of S.

The cell sorting of fluorescence-activation (FACS) can be used to sort the cell of expressing CD8 by the antibody of cell with suitable mark is contacted.In one embodiment, other antibody and FACS letter sorting also can be used for producing pure basically CD8 ⁺CD3 ⁺Cell colony, or be used for the cell that purifying is not expressed detectable CD4 or CD56 level.

Among complicated detection level, FACS has utilized a plurality of Color Channels, low angle and obtuse angle light scattering sense channel to separate or sorting cell with impedance channel.Can use any FACS technology, as long as it is harmless to the viability of target cell.(for the illustrative methods of FACS, referring to U.S. Patent No. 5,061,620, its this draw be with reference to).Similarly, FACS can be used for purifying CD8 basically ⁺Cell is for example expressed CD3 but is not expressed the CD8+ cell of CD56 or CD4.

But, can utilize the technology of other different usefulness to come purifying and separate required cell colony.Employed isolation technics should keep the viability of cell grade part to be collected the biglyyest.Certainly, employed concrete technology depends on the efficient of separation, the cytotoxicity of method, the easiness of separation and the equipment and/or the technical skill of speed and needs.

Separable programming can comprise the magnetic beads of using antibody sandwich magnetic resolution, affinity chromatography, link to each other with monoclonal antibody or the cytotoxicity medicament that is used in combination with complement and utilize monoclonal antibody or other " elutriations " that makes things convenient for technology that is attached on the solid-phase matrix.Be attached to for example antibody of sepharose 4B, polystyrene bead, hollow-fibre membrane and plastics petri diss of magnetic beads and other solid-phase matrix, allow directly to separate., can from cell suspension, remove solid support and cell suspension physical separation by simply by the cell of antibodies.Cell is connected the accurate condition and the time span of antibody incubation with solid phase, depend on use system specific several factors.Yet the felicity condition of selection is within the technical scope in present technique field.

Then, after the cell that allows expression blip thing (for example CD8) has the antibody of time enough in conjunction with the solid phase connection, can or wash unconjugated cell off with the physiological buffer wash-out.Depend primarily on the character of employed solid phase and antibody then, by the cell of any suitable method separating and combining on the solid phase.

Antibody can engage with biotin, can use Avidin or streptavidin that combines with holder or the fluorophore that uses the cell sorting device (FACS) that can be used for fluorescence-activation then, is removed, can carry out cell separation (referring to above).At the beginning, can cd8 cell and other cell separation be opened by the cell surface expression of CD3.In a concrete non-limiting instance, separate CD3 by magnetic beads ⁺Cell carries out the positive to be selected, and wherein magnetic beads is coated with the reactive monoclonal antibody of CD3.Then can be with CD3 ⁺Cell takes off from magnetic beads.

CD3 ⁺Cell can be realized by cultivating release or additive method from the release on the magnetic beads.Then, if necessary, for example use

(Becton Dickinson, San Jose CA) check the CD3 that is separated to flow cytometer ⁺The purity of cell.In one embodiment, carry out further purification step, for example the FACS letter sorting of the cell colony that discharges from magnetic beads.

In one embodiment, use magnetic beads to separate at first to separate and do not express for example cell colony of B220, CD4, CD45, CD5 or CD56 of more than one pedigree specificity marker things.In addition, can use elutriation to separate and not express the cell of one or more B cells or macrophage pedigree specificity marker thing (for the elutriation method, referring to Small etc., J Immunol Methods3; 167 (1-2): 103-7,1994, its this draw be with reference to).

In several embodiments, in case after separating, with CD8 ⁺The T cell descended external incubations 2 to 9 days, for example about 4 days with Mtb polypeptide or its fragment that combines MHC at 37 ℃.In several examples, comprise Mtb polypeptide or its fragment in conjunction with MHC (its concentration for for example about 5 to about 25 μ g/ml, for example about 5, about 10, about 15 or about 20 μ g/ml).In several examples, can be with another aliquot incubation under the situation that does not have the Mtb polypeptide of T cell sample, with comparing.Also can utilize more than one Mtb polypeptide.

In one embodiment, from sample separation monocyte (MC).MC comprises T cell and antigen presenting cell (APC).Therefore in method, the APC that exists among the MC that is separated to can be presented to peptide the T cell.In another embodiment, have only the T cell, for example have only CD8 ⁺The T cell can be from sample purifying.

The APC that uses in method can be any cell that has I class MHC molecule in its surface.It can be or can not be the antigen presenting cell of specialization, for example B cell, dendritic cell or macrophage.The APC that uses in method can come from the host identical with the T cell.In general, APC can be to T presented by cells peptide.APC can be the isolated cells or the cultured cells of fresh separated, for example from the cell of clone.APC can be allochthonous or from body.

The T cell that stems from the destination object sample can be placed to have all Mtb polypeptide (or the merging thing of Mtb polypeptide or specific Mtb polypeptide) and be intended for use to test the analysis of relevant group collection, perhaps can and place the different analysis that respectively contains one or more peptides T cell portioning.In one embodiment, one or more polypeptide with the shown amino acid sequence of SEQ ID NO:1-12, SEQ ID NO:39 or SEQ ID NO:61 or the fragment in conjunction with MHC of one or more these polypeptide have been used.In other embodiments, one or more described polypeptide are ESAT6 or CFP10, but any Mtb polypeptide can use.Other peptides that use show in SEQ ID NO:39-83.Can use any two or more Mtb peptides disclosed herein, with simultaneously, respectively or the T cell of these polypeptide of identification that use in order.Can use other combinations of any Mtb polypeptide disclosed herein.Also can use the merging thing of Mtb polypeptide.

In one embodiment, under the situation that does not have the T cell, provide one or more peptides to being delivery cell.Then, typically after allowing on this cell surface, to present peptide, provide it to the T cell that separates from object.

The time span of peptide and cells contacting will become according to the method for the identification that is used for definite peptide.In typical case, analyze adding 10 to each ⁵To 10 ⁷, about 5X10 for example ⁵To 10 ⁶Individual T cell.Analyzing under the situation that directly adds peptide to this, its concentration is typically about 10 ^-1To about 10 ³μ g/ml, for example about 0.5 to about 50 μ g/ml or about 1 to about 10 μ g/ml.The time span of T cell and peptide incubation can be from about 4 to about 24 hours, and for example about 6 to about 16 hours, or about 12 hours.

Determine that peptide is by T cell CD8 for example ⁺The identification of T cell-specific can be carried out with combining of T cell by measuring peptide.In typical case, the T cell of binding peptide can sort according to this combination, for example uses cell sorting (FACS) technology (referring to above) of fluorescence-activation.If use the frequency of the cell that peptide is sorted to be higher than control value, can think the existence of T cell that identification polypeptide has taken place to detect.

Determine whether identification polypeptide of T cell, also can be by whether binding peptide carries out having the variation that detects the T cell state under the situation of peptide or definite T cell.The variation of state is generally caused by the antigentic specificity functional activity of T cell behind the TXi Baoshouti binding peptide.In general, after in conjunction with TXi Baoshouti, peptide is incorporated on the I class MHC molecule, and it can be presented on PBMC or antigen presenting cell (APC) surface.

The T cell activation can detect by the known any means of the professional in present technique field.In an example, CD8 ⁺The T cell activation detects by assessing molten cytoactive.In another example, CD8 ⁺The T cell activation detects by breeding.In several examples, with do not infect object in compare the propagation level of high at least twice and/or high at least 20% lysis level, show destination object for example children, suffer from the object of LTBI and have mycobacterial infections.In other examples, with do not infect object in compare the propagation level of high at least twice and/or molten born of the same parents' level of high at least 20%, show that object suffers from extrapulmonary tuberculosis and/or suffer from pulmonary tuberculosis.Object can be any destination object, for example children.

The variation of T cell state can be from the material of T cell cell factor such as interferon (IFN)-γ, IL-2 or TNF-α begins secretion or secretion increases for example.In an example, can detect this material by the existence that allows material to combine, measure then specificity combinating reagent/substance complex with specificity combinating reagent.Specificity combinating reagent is typically antibody, for example with the material polyclone or the monoclonal antibody that combine of cell factor for example.Antibody at cell factor is commercially available, maybe can use standard technique manufacturing.

In typical case, with specificity combinating reagent for example antibody immobilization on solid support.After allowing the cell factor combination, can choose wantonly and clean solid support to remove the material that does not combine with antibody specificity.Antibody/cell factor compound can be by using second kind of binding reagents combining with compound, for example using the antibody of label mark (directly or indirectly) to detect.In general, second kind of reagent is different with the site that combines first kind of reagent with the site of material combination.

In several examples, second kind of binding reagents can detect by the third reagent with the direct or indirect mark of detectable.For example, second kind of reagent can comprise biotin, allow involved streptavidin and label for example the third reagent of enzyme labeling thing, radioactivity label or fluorescent marker detect.

In one embodiment, detection system is that ELISPOT analyzes, and for example announces the analysis of describing among No.WO 98/23960 or the U.S. Patent application No.2005/0208594 at PCT, and the two draws at this and is reference.In an example, be immobilized in first kind of IFN-γ specific antibody combination on the solid support from the IFN-γ of T emiocytosis.Use the IFN-γ of second kind of IFN-γ specific antibody detection combination of detectable label substance markers then.Exemplary labelled antibody is commercially available, for example from MABTECH ^TM(Stockholm, SWE).Exemplary ELISPOT analytic approach is described in the following examples part.Detection method can be any other method that is used to detect cytokine-expressing, and referring to the european patent application No.EP1867988 that has for example announced, it draws at this and is reference.

The variation of the T cell state that can also measure can be for example increase of thymine absorption of material that the T cell is taken in.The variation of cell surface marker is measured on the increase that the variation of state also can be by the T cell size or the propagation of T cell or the T cell.

This paper provides the cell (CD8 that combines with Mtb polypeptid specificity disclosed herein, be used to detect expression CD8 ⁺) reagent.These reagent are tetramer I class MHC/ immunogenicity TARP polypeptide complexes.These tetramer compounds comprise the Mtb polypeptide, and for example specificity is 9 to 30 amino acid whose polypeptide in conjunction with the length of I class MHC.

Tetramer I class MHC/ peptide complexes can use method well-known in the art synthesize (Altmann etc., Science 274:94,1996, its this draw be with reference to).In a concrete limiting examples, can utilize prokaryotic expression system to come the HLA heavy chain polypeptide and the B2M (β 2m) of synthesizing and purifying.A concrete limiting examples of the expression system that uses is (the R﹠amp of pET system; D Systems, Minneapolis, MN).Stride film and cytoplasmic tail and, heavy chain is modified by deletion in the terminal sequence of adding the enzyme process biotinylation site of containing biotin protein ligase (Bir-A) of COOH.Then that heavy chain, β 2m and peptide is folding again.Again Zhe Die product can separate by any means known in the art, carries out biotinylation by Bir-A then.By biotinylated product is contacted with streptavidin, produce the tetramer then.

In one embodiment, streptavidin is labeled.The label that is fit to includes but not limited to enzyme, magnetic beads, colloidal state magnetic beads, haptens, fluorophore, metallic compound, radioactivity compound or medicine.The enzyme that can engage with streptavidin includes but not limited to alkaline phosphatase, peroxidase, urase and beta galactosidase.The fluorophore that can engage with streptavidin includes but not limited to fluorescein isothiocyanate, tetramethyl rhodamine isothiocyanates, phycoerythrin, allophycocyanin and texas Red.For other fluorophores that can engage with streptavidin, referring to Haugland, R.P. " molecular probe: fluorescence probe and research with the chemical substance handbook (Molecular Probes:Handbook of Fluorescent Probes and Research Chemicals) (1992-1994).The metallic compound that can engage with streptavidin includes but not limited to ferritin, collaurum and particularly colloid superparamagnetism pearl.The haptens that can engage with streptavidin includes but not limited to biotin, digitophyllin, azolactone and nitrophenols.The radioactivity compound that can engage with streptavidin is known in the present technique field, and include but not limited to Tc 99m ( ⁹⁹Tc), ¹²⁵I and comprise any radioactive nuclide and include but not limited to ¹⁴C, ³H and ³⁵The amino acid of S.In general, in method disclosed herein, use the streptavidin of fluorophore mark.

In one embodiment, produced the cell suspension of the T cell that comprises specific recognition Myb polypeptide, and cell has been reacted with the described tetramer in suspension.In one embodiment, these reagent are used to labeled cell, analyze by cell sorting (FACS) pair cell of fluorescence-activation then.The machine that is used for FACS has utilized a plurality of Color Channels, low angle and obtuse angle light scattering sense channel to separate or sorting cell with impedance channel among complicated detection level.Can use any FACS technology, as long as it is harmless to the detection of target cell.(for the illustrative methods of FACS, referring to U.S. Patent No. 5,061,620, its this draw be with reference to).

Be used to detect the method that Mtb infects: the skin test checking

On the other hand, except top disclosed use CD8 ⁺Outside the method for T cell, use skin test to verify that property testing is to confirm mycobacterial infections, particularly diagnosis lungy." skin test " is any analytical approach of directly carrying out on one's body the patient, wherein after above-described polypeptide is administered in the skin for example intracutaneous injection with one or more, measures delayed super quick (DTH) reaction (for example scleroma, swelling, rubescent or dermatitis).Such injection can use any suitable device, for example tuberculin syringe or the 1ml syringe that are enough to polypeptide is contacted with patient's dermal cell to realize.In several examples, be reflected at after the injection at least 48 hours, for example measuring between about 48 hours to about 72 hours after the injection.

DTH reaction is a cell-mediated immune responses, it is former be exposed in the object of test antigen (Mtb polypeptide, its fragment or its fusion) in conjunction with MHC stronger.Replying can vision measurement, for example uses ruler.In several examples, diameter is positive response greater than about 0.5cm, for example diameter greater than replying of about 1.0cm, and has shown mycobacterial infections.

The Mtb polypeptide that uses in skin test can be mixed with and contain the pharmaceutical composition that polypeptide and physiology can be accepted carrier.These compositions typically comprise one or more Mtb polypeptide (or it is in conjunction with fragment or its fusion of MHC), and its amount about 1 μ g in the 0.1ml volume arrives about 100 μ g, for example about 10 μ g arrive in the scope of about 50 μ g.The carrier that uses in pharmaceutical composition can be to have suitable antiseptic for example phenol and/or TWEEN80 ^TMBrine solution.

In general, the polypeptide that uses in skin test has enough sizes, makes it be retained in the injection site place during the reaction time.In several examples, just it is enough at least 9 amino acid whose polypeptide for length.Without being limited by theory, polypeptide is decomposed by macrophage in several hours after injection, to allow to be presented to the T cell.Such polypeptide can comprise the repetitive sequence of one or more top disclosed sequences and/or other immunogenicities or non-immunogenic sequence.

Therefore, determine that peptide can be measured in vivo by the T cell recognition.In several examples, peptide is delivered medicine to individuality, then can meter understand the replying of identification of peptide.In one embodiment, peptide is typically to test similar mode intradermal administration to Mantoux.Peptide can the epidermis administration.Peptide typically by syringe needle for example by drug administration by injection, but also can for example impact (ballistics), for example be used for the impact technology administration of nucleic acid delivery by additive method.The EPC application No.EP-A-0693119 that has announced has described the technology that can typically be used for the peptide administration.In several examples, administration 0.001 to 1000 μ g, for example 0.01 to 100 μ g or 0.1 to 10 μ g peptide.The medicament that peptide alternatively, can administration can be provided in vivo.Therefore, the polynucleotide that can administration can express described polypeptide.Polynucleotide typically have the characteristic of any polynucleotide of discussing below.Polypeptide is expressed from polynucleotide in vivo, and the identification that can measure peptide in vivo.In typical case, administration 0.001 to 1000 μ g, for example 0.01 to 100 μ g or 0.1 to 10 μ g polynucleotide.

Be used to detect the method that Mtb infects: checking property testing, antibody test

On the other hand, except top disclosed use CD8 ⁺Outside the method for T cell, in analysis, use one or more polypeptide to carry out the checking property testing, to determine in the biological sample (such as but not limited to whole blood, sputum, serum, blood plasma, saliva or cerebrospinal fluid) at the antibody of polypeptide with respect to the existence of contrast or do not exist.To the sensitization of mycobacteria antigen, it can show mycobacterial infections and particularly tuberculosis before the existence of this antibody showed.

In the embodiment of using more than one polypeptide, polypeptide can be complementary, makes a kind of composition polypeptide with the infection in the test sample, and wherein said infection can not be detected by another kind of composition polypeptide.Complementary polypeptide generally can be identified by using every peptide species to assess from a series of known blood serum samples that obtained by the patient of mycobacterial infections individually.After determining the every peptide species of use correctly is accredited as the positive with which sample, can make the combination that can in great majority or all tested samples, detect two or more polypeptide that infect.Complementary polypeptide can be used for increasing the sensitivity of diagnostic test.The above-mentioned Mtb polypeptide that therefore, in analysis, can comprise more than one.Can choose wantonly from other polypeptide of Mtb (not describing in this article) and to be included in the analysis.

There is multiple assay format to can be used for antibody in the test sample (referring to " antibody experiment guide " (Antibodies:A Laboratory Manual) of for example Harlow and Lane, Cold Spring Harbor Laboratory (1988), its this draw be with reference to).In general, the existence that Mtb infects among the patient or do not exist can be definite as getting off: (a) will contact with one or more Mtb polypeptide from the biological sample that the patient obtains; (b) the existing of the antibody that combines with polypeptide in the test sample (or not existing); And (c) antibody horizontal and contrast are compared.Contrast can be a standard value, for example Yu Ding cutoff.Contrast can be the amount of antibody in the known object that is infected by Mtb, or in the known object that is not infected by Mtb the amount of the antibody of specific binding polypeptide.

In several embodiments, analytical approach comprises uses the polypeptide that is immobilized on the solid support.The antibody of specificity combining target polypeptide combines with solid support.Can use the detectable that comprises detectable to detect the antibody of combination then.The detectable that is fit to comprises the antibody of the mark that combines with antibody/polypeptide complex.The detectable that is fit to also comprises second kind of unlabelled antibody combining with the antibody polypeptides compound and specificity the 3rd antibody in conjunction with second antibody.The detectable that is fit to also comprise with the reporter group mark not in conjunction with polypeptide (for example in half competitive analysis method).

Alternatively, can use the competitive analysis method, the antibody and the sample incubation that wherein will combine, use the reporter group mark with target polypeptides.Behind incubation, after with immobilized antigen and sample incubation, allow antibody to combine with immobilized antigen.The component of sample suppresses the degree that combines of labelled antibody and immobilization polypeptide, has shown the reactivity of sample and immobilization polypeptide.

The solid support that uses in analytic approach disclosed herein can be any solid material that can adhere to antigen.For example, solid support can be instrument connection or cellulose nitrate or other film that is fit in the microtiter plate.Alternatively, solid support can be pearl or disk, for example for example polystyrene or Polyvinylchloride of glass, glass fibre, latex or plastic material.Holder also can be magnetic particle or optical fibre transducer, and is for example in U.S. Patent No. 5,359, disclosed in 681.

Can use various technology that polypeptide is attached on the solid support.The combination of polypeptide can by non-covalent association for example adsorb or covalent attachment for example directly connecting key or connecting key by crosslinking chemical between the functional group on antigen and the holder realizes.

For combination, in conjunction with realizing by one or more Mtb polypeptide (generally in damping fluid) are contacted an amount of time with solid support by absorption.At combination duration of contact the typical case between about 1 hour to 1 day.In general, in conjunction with realizing by polystyrene or Polyvinylchloride solid support are contacted with one or more Mtb polypeptide, the amount of described Mtb polypeptide at about 10ng in the scope of about 1 μ g, for example about 100ng antigen.

The covalent attachment of target Mtb polypeptide and solid support generally can by with holder with can for example hydroxyl or the amino bifunctional reagent that all responds react and realize with functional group on holder and the polypeptide.For example, can use benzoquinones or the condensation by the aldehyde radical on the holder and amine on the polypeptide and reactive hydrogen, the Mtb polypeptide is attached to (" Pierce immunological technique catalogue and handbook (Pierce Immunotechnology Catalog and Handbook) on the holder with suitable polymer coating, A12A13,1991).

In certain embodiments, analysis is Enzyme Linked Immunoadsorbent Assay (ELISA).This analysis can contact with sample and carries out by at first will being immobilized on the solid support polypeptide antigen in (for example in the hole of microtiter plate), and the mode of described contact makes the antibody of the specificity combining target polypeptide that exists in the sample combine with immobilized polypeptide.Remove unconjugated sample then, and add the detectable that can combine with immobilized antibody-polypeptide compound.The method that use is suitable for concrete detectable is measured the amount of the detectable that keeps combination.For example, detection method can detect the existence of fluorescence or enzymatic activity.

In certain embodiments, polypeptide is immobilized on the holder; The typical case is with any residual protein binding site blocking-up on the holder.Can use any suitable blocking agent to block unconjugated protein binding site, for example can use bovine serum albumin(BSA) or TWEEN 20 ^TMThen with immobilized polypeptide and sample incubation, and allow antibody to combine with antigen.Before incubation, can be with sample with for example salt solution (PBS) dilution of damping fluid such as phosphate-buffered of the thinning agent that is fit to.In general, be the time span that is enough to detect the existence of antibody in the sample of mycobacterial infections the duration of contact of Shi Heing (incubation time).In a concrete non-limiting instance, be enough to duration of contact to obtain in conjunction with unconjugated antibody between at least 95% of the level that combines that reaches when being in balance.Reaching the required time of balance can be by analyzing determining in conjunction with level of taking place in a period of time.At room temperature, in general about 30 minutes incubation time is enough.

Then can be by with the damping fluid that is fit to, for example contain 0.1%TWEEN 20 ^TMPBS washing solid support, remove not in conjunction with sample.Can add detectable to solid support then.Detectable can be the compound that any and immobilized antibody-polypeptide compound combines and can be detected.In several embodiments, detectable comprises the binding reagents (for example albumin A, Protein G, immunoglobulin (Ig), agglutinin or free antigen) that engages with label.The label that uses comprises enzyme (for example horseradish peroxidase), substrate, co-factor, inhibitor, dyestuff, radioactive nuclide, luminophore, fluorophor and biotin.Bond can use methods known in the art to realize with engaging of label; The binding reagents that engages also be commercially available (for example from Zymed Laboratories, San Francisco, Calif. and Pierce, Rockford, Ill.).

The time quantum that detectable and immobilized antibody-polypeptide composition incubation is enough to detect the antibody that combines.The time quantum that is fit to generally can be determined from the instructions of manufacturer or by the level of analyzing the combination that takes place in a period of time.Remove unconjugated detectable then, and the usage flag quality testing is surveyed the detectable of combination.For radioactively labelled substance, can use scintillation counting or radioautograph method to detect.Can use spectrographic technique to detect dyestuff, luminophore and the fluorophor that is used as label.Biotin can use and for example affine detection usually of radioactively labelled substance, fluorescent marker or the coupling of enzyme labeling thing of different labels.The enzyme labeling thing can be by adding substrate (generally carrying out specific time span), then reaction product being carried out spectrum or other and analyze and detect.

For the existence of determining anti-bacillus antibody in the sample or do not exist, generally will from label that solid support combines on detected signal and contrast compare.In one embodiment, contrast is a standard value, for example when the average signal that immobilized antigen is obtained with from the sample incubation of infected patient not the time.In general, produce the sample be higher than the signal that contrasts two or three standard deviations, be considered to the mycobacterial infections positive.In another embodiment, control value uses recipient's working curve (Receiver Operator Curve), " clinical epidemiology: clinical medical basic science " (Clinical Epidemiology:A Basic Science for Clinical Medicine) according to Sackett etc., Little Brown and Co., the method for describing in the 1985 106-107 pages or leaves is determined.In simple terms, in this embodiment, from True Positive Rate (sensitivity) and the definite control value of false positive rate (100% specificity) pairing figure that may control value corresponding to each of diagnostic test results.The control value that surrounds maximum area on figure is the most accurate cutoff, produces the sample of the signal be higher than the cutoff of determining by this method, is considered to positive.Alternatively, can mobile cutoff to minimize false positive rate or to minimize false negative rate.In general, produce the sample of the signal be higher than the cutoff of determining by this method, be considered to the tuberculosis positive.

In related embodiment, described analysis to be to flow through fast or the test-strips form carries out, and wherein antigen is immobilized in film, such as but not limited on the cellulose nitrate.In flowing through test, when sample process film, the antibody in the sample combines with immobilized polypeptide.When the flow of solution that contains detectable was crossed film, detectable (for example albumin A-collaurum) combined with the antibody-polypeptide compound.Detecting the detectable of combination can carry out as mentioned above.

In an example of test-strips form, an end that film is combined with polypeptide is immersed in the solution that contains sample.Sample moves along film, by the zone of containing detectable and the zone that arrives the immobilization polypeptide.The concentration of polypeptide place detectable has shown the existence of anti-bacillus antibody in the sample.In typical case, the concentration of this site detectable produces for example line of the pattern can vision read.Do not exist this pattern to represent negative findings.In general, the amount that is immobilized in the polypeptide on the film is selected, when containing the antibody horizontal that is enough to generation positive signal in Enzyme Linked Immunoadsorbent Assay analysis (ELISA) with convenient biological sample, but the pattern of generation visual discrimination.In several embodiments, the amount that is immobilized in the polypeptide on the film arrives about 1 μ g at about 25ng, for example about 50ng arrives in the scope of about 500ng.Such test is typically used very, and the patients serum or the blood of small size carry out.

Detect the method that Mtb infects: the checking property testing that is used to detect polynucleotide

On the other hand, except top disclosed use CD8 ⁺Outside the method for T cell, the existence by the mRNA of coding mycobacterium polypeptide in the detection of biological sample, do not exist or level, carried out the checking property testing.In several examples, utilized hybridization analysis, for example the Northern marking or the spot marking are analyzed.In other examples, utilized the analysis of PCR-based.

The universal method that is used for the mRNA extraction is being known in the art, and be disclosed in the molecular biology textbook of standard, " molecular biology modernism " (the Current Protocols of Molecular Biology) that comprises Ausubel etc., John Wiley and Sons (1997).Be used for being disclosed in for example Rupp and Locker from the method for paraffin-embedded tissue extraction RNA, Lab Invest.56:A67 (1987), and De Andres etc., among the BioTechniques 18:42044 (1995).Specifically, RNA separates and can from commercial manufacturers for example use Purification kit, damping fluid group and proteinase, carry out according to the instructions of manufacturer.For example, the total RNA from the cell the culture (for example obtaining from object) can use

Small-sized post separates.Other commercially available RNA separating kits comprise

Full DNA and RNA purification kit (

Madison, Wis.) and paraffin mass RNA separating kit (Ambion, Inc.).Total RNA from tissue sample can use RNA Stat-60 (Tel-Test) to separate.Can separate by for example cesium chloride density gradient centrifugation from the RNA of biological sample preparation.

Being used for the mRNA quantitative methods is being known in the art.In an example, method has been utilized reverse transcriptase polymerase chain reaction (RT-PCR).In general, be that the reverse transcription of RNA template is become cDNA in first step of being undertaken by RT-PCR in the expression conditions analysis, in the PCR reaction, it is carried out the index amplification then.Two kinds of the most frequently used reverse transcriptase are avian meloblastosis virus reverse transcriptase (AMV-RT) and Moloney (Moloney) murine leukemia virus reverse transcriptase (MMLV-RT).Reverse transcription step typically uses Auele Specific Primer, random hexamer or oligomerization dT primer to cause according to the target of environment and expression analysis.For example, the RNA of extraction can use GeneAmp RNA PCR kit (Perkin Elmer, Calif. USA), carry out reverse transcription according to the instructions of manufacturer.The cDNA that produces can be reacted as the PCR that template is used for subsequently then.

Although the PCR step can be used various heat-staple DNA dependent dna-polymerases, its typical case's use has the Taq archaeal dna polymerase that 5 '-3 ' nuclease still lacks 3 '-5 ' proofreading endonuclease enzymatic activity.Therefore,

PCR typically utilize 5 of Taq or Tth polymerase '-hybridization probe that nuclease comes hydrolysis to combine with the target amplicon, but also can use any enzyme with 5 ' nuclease of equal value.Use two Oligonucleolide primers to produce PCR and react typical amplicon.The 3rd oligonucleotides or probe are designed to detect the nucleotide sequence between two PCR primers.Probe can not be extended by the Taq archaeal dna polymerase, and with reporting fluorescent dye and cancellation fluorochrome label.When two kinds of dyestuffs when the position is close together on probe, from the emission of any induced with laser of reporting dyes by the quencher dyes cancellation.During amplified reaction, the Taq archaeal dna polymerase cuts probe in template dependence mode.The probe fragment that obtains dissociates in solution, and is not subjected to the influence of the quenching effect of second fluorophore from the signal of the reporting dyes that discharges.For each synthetic recruit, discharge a reporter dye molecules, and the detection of the reporting dyes of cancellation is not that the quantitative interpretation of data provides the foundation.

RT-PCR can use commercially available equipment to carry out, for example ABIPRISM

Sequence detection system ^TM(Perkin-Elmer-Applied Biosystems, Foster City, Calif., USA) or Lightcycler (Roche Molecular Biochemicals, Mannheim, Germany).In one embodiment, 5 ' nuclease program is at real-time quantitative PCR device ABI PRISM for example

Sequence detection system

Last operation.System comprises thermal cycler, laser instrument, charge-coupled device (CCD) (CCD), camera and computing machine.System is with the 96 orifice plate forms sample that increases on thermal cycler.During increasing, the fluorescence signal of the induced with laser by all 96 holes of Connectorized fiber optic cabling real-time collecting, and on CCD, detect.System comprises the software that is used to move instrument and analyzes data.

In some instances, 5 '-nuclease analyzes data and at first is expressed as Ct or threshold cycle.As discussed above, at each record fluorescent value, and be illustrated in product amount when increasing this in the amplified reaction with it cycle period.Point when recording the significant fluorescence signal of statistics first is threshold cycle (Ct).

For sum of errors influence that will change between the sample drops to minimumly, mark carries out RT-PCR in can using.Be marked in desirable between the different tissues and express, and do not tested the influence of processing with constant level.Be most commonly used to the normalized RNA of gene expression pattern is the mRNA and the 18S rRNA of house-keeping gene glyceraldehyde-3-phosphate-dehydrogenasa (GAPDH), beta-actin.

The nearest variation of RT-PCR technology is a real-time quantitative PCR, and its product fluorescence probe by double labeling (promptly

Probe) measures the accumulation of PCR product.PCR in real time is carried out normalized quantitative competitive PCR with the internal competition thing that uses each target sequence and is included in the normalization gene in the sample with use or is used for the quantitative comparison PCR of house-keeping gene of RT-PCR compatible (referring to Held etc., Genome Research 6:986994,1996).Quantitative PCR also is described in U.S. Patent No. 5,538, and in 848, its disclosure is drawn at this and is reference.Relevant probe and quantitative amplification program description be in U.S. Patent No. 5,716,784 and U.S. Patent No. 5,723,591 in, its disclosure is drawn at this and is reference.Being used for can be from PE Applied Biosystems at the instrument of microtiter plate execution quantitative PCR, 850 Lincoln Centre Drive, and Foster City, Calif.94404 obtains, trade name ABI

7700.

Use fixing paraffin-embedded tissue as the RNA source, the step of gene expression being carried out quantitative representative solution comprises mRNA separation, purifying, primer extension and amplification, they provide in the magazine article of various publication (referring to Godfrey etc., J.Molec.Diagnostics 2:8491,2000; K.Specht etc., Am.J.Pathol.158:41929,2001).In simple terms, representational method is from cutting out the thick paraffin-embedded tissue sample section of about 10 μ m.Extract RNA then, remove deproteinize and DNA.After analyzing RNA concentration, can comprise RNA if desired and repair and/or amplification step, and use the gene specific promoter then by RT-PCR with the RNA reverse transcription.

Optionally the quantitative nucleic acid amplification program description is in U.S. Patent No. 5,219, and in 727, it draws at this and is reference.In this program, determine the amount of target sequence in the sample by while amplified target sequence and interior mark nucleic acid segment.Mensuration is from the amount of the DNA of each section amplification, and compares amount with the target nucleic acid section of determining to exist in the sample before the amplification with typical curve.

In some embodiment of this method, also can assess the expression of " looking after the house " gene or " internal contrast (internal control) ".These terms mean and comprise that its existence can assess any composition of mRNA level of cell factor or the gene of comprehensive representation.Such assessment comprises become second nature level and be used for the contrast of the variation that RNA reclaims of the Overall Group that measures genetic transcription.

The development that monitoring is infected and/or the validity of therapy

In several embodiments, diagnostic method disclosed herein is used for for example children or suffers from the development of the object monitoring mycobacterial infections of LTBI.In this embodiment, can in a period of time, carry out the aforesaid analytical approach that is used for diagnosis of mycobacterial infections, and measure CD8 ⁺The variation of t cell responses.For example, analysis can be carried out then as required in for example execution in per approximately 12,24,36,48,60 or 72 hours in several months or several weeks of the time period of appointment.

In general, for example using for example CD8 that arrives of the detection of expression of IFN-γ of cell factor ⁺Among those patients that the reactivity of T cell increases in time, mycobacterial infections develops.On the contrary, work as CD8 ⁺The reactivity of T cell keeps constant in time or when reducing, mycobacterial infections is development not.By this way, can or suffer from the validity of assessment particular treatment in the object of LTBI for example children.

In one embodiment, object for example T cell, for example CD8 of specific recognition Mtb polypeptide among the children have been assessed ⁺T cell and/or CD4 ⁺The existence of T cell.Object is implemented therapeutic scheme.Assess the existence of the T cell of specific recognition Mtb polypeptide then.Implement the CD8 of specific recognition Mtb polypeptide before with therapeutic scheme ⁺The amount of T cell is compared, the CD8 of corresponding specific recognition Mtb polypeptide ⁺The reduction of the amount of T cell or constant shows that therapeutic scheme is invalid.Implement the CD8 of specific recognition Mtb polypeptide before with therapeutic scheme ⁺The amount of T cell is compared, the CD8 of specific recognition Mtb polypeptide ⁺The increase of the amount of T cell shows that therapeutic scheme is effective.Also can measure CD4 ⁺Cell.

Be noted that for any above-mentioned analytical approach,, can analyze a plurality of mycobacterium marks in the given sample in order to improve sensitivity.Obviously, analytical approach disclosed herein can be used in combination.Therefore, mycobacterium polypeptide group, and the combination of analytical approach can be used for optimizing sensitivity and specificity.

The mycobacterium polypeptide

Disclose several mycobacterium polypeptide herein and can be used for inducing immune response at Mtb, for example t cell response.The mycobacterium polypeptide can be used in the diagnostic analysis, to identify to infect for example object of Mtb of mycobacterium is arranged.In several embodiments, polypeptide comprises the amino acid sequence of following demonstration or is made of it:

1.MX1SRFMTDPHAMRDMAGRFEVHAQTVEDEARRMWASAQNISGAGWSGMAEATS LDTMX ₂X ₃MNQAFRNIVNMLHGVRDGLVRDANNYEQQEQASQQILS, (SEQ ID NO:1, wherein X1 is A or T, X ₂Be T or A, and X ₃Be any amino acid, for example Q or do not have amino acid)

In several examples, polypeptide comprises the amino acid sequence of following demonstration or is made of it basically or is made of it:

A.MASRFMTDPHAMRDMAGRFEVHAQTVEDEARRMWASAQNISGAGWSGMAEATSL DTMTQMNQAFRNIVNMLHGVRDGLVRDANNYEQQEQASQQILS (SEQ ID NO:2) (the also TUBERCULIST No.Rv1038c that provides referring on March 1st, 2007, draw at this and to be reference, be called as EsxJ, ES6_2, TB11.0, QILSS)

B.MASRFMTDPHAMRDMAGRFEVHAQTVEDEARRMWASAQNISGAGWSGMAEATSL DTMAQMNQAFRNIVNMLHGVRDGLVRDANNYEQQEQASQQILSS (SEQ ID NO:3, TUBERCULIST No.Rv1197, on March 1st, 2007 provided, draw at this and to be reference, be also referred to as EsxK, TB11.0, QILSS)

C.MASRFMTDPHAMRDMAGRFEVHAQTVEDEARRMWASAQNISGAGWSGMAEATSL DTMT+MNQAFRNIVNMLHGVRDGLVRDANNYEQQEQASQQILSS (SEQ ID NO:4, TUBERCULIST No.Rv1992, on March 1st, 2007 provided, draw at this and to be reference, be called as EsxM, TB11.0, QILSS).

D.MATRFMTDPHAMRDMAGRFEVHAQTVEDEARRMWASAQNISGAGWSGMAEATSL DTMAQMNQAFRNIVNMLHGVRDGLVRDANNYEQQEQASQQILSS (SEQ ID NO:5, TUBERCULIST No.Rv2347c, on March 1st, 2007 provided, draw at this and to be reference, be also referred to as EsxP, ES6_7, QILSS)

E.MTSRFMTDPHAMRDMAGRFEVHAQTVEDEARRMWASAQNISGAGWSGMAEATSL DTMTQMNQAFRNIVNMLHGVRDGLVRDANNYEQQEQASQQILSS (SEQ ID NO:6, TUBERCULIST No.Rv3620c, on March 1st, 2007 provided, draw at this and to be reference, be also referred to as EsxW, ES6_10, QILSS).

In other embodiments, polypeptide comprises the amino acid sequence of following demonstration or is made of it basically or is made of it:

2. MSYMIATPAALTAAATDIDGIGSAVSVANAAAVAATTGVLAAGGDEVLAAIARLFNANAEEYHALSAQVAAFQTLFVRTLTGGCGVFRRRRGRQCVTAA

3.VSLVIATPQLLATAALDLASIGSQVSAANAAAAMPTTEVVAAAADEVSAAIAGLFGAHARQYQALSVQVAAFHEQFVQALTAAAGRYASTEAAVERSLLGAVNAPTEALLGRPLIGNGADGTAPGQPGAAGGLLFGNGGNGAAGGFGQTGGSGGAAGLIGNGGNGGAGGTGAAGGAGGNGGWLWGNGGNGGVGGTSVAAGIGGAGGNGGNAGLFGHGGAGGTGGAGLAGANGVNPTPGPAASTGDSPADVSGIGDQTGGDGGTGGHGTAGTPTGGTGGDGATATAGSGKATGGAGGDGGTAAAGGGGGNGGDGGVAQGDIASAFGGDGGNGSDGVAAGSGGGSGGAGGGAFVHIATATSTGGSGGFGGNGAASAASGADGGAGGAGGNGGAGGLLFGDGGNGGAGGAGGIGGDGATGGPGGSGGNAGIARFDSPDPEAEPDVVGGKGGDGGKGGSGLGVGGAGGTGGAGGNGGAGGLLFGNGGNGGNAGAGGDGGAGVAGGVGGNGGGGGTATFHEDPVAGVWAVGGVGGDGGSGGSSLGVGGVGGAGGVGGKGGASGMLIGNGGNGGSGGVGGAGGVGGAGGDGGNGGSGGNASTFGDENSIGGAGGTGGNGGNGANGGNGGAGGIAGGAGGSGGFLSGAAGVSGADGIGGAGGAGGAG GAGGSGGEAGAGGLTNGPGSPGVSGTEGMAGAPG ( SEQID NO：8； TUBERCULIST No.Rv2487; On March 1st, 2007 provided; Draw at this and to be reference, be also referred to as PE_PGRS42)

4.MHQVDPNLTRRKGRLAALAIAAMASASLVTVAVPATANADPEPAPPVPTTAASP PSTAAAPPAPATPVAPPPPAAANTPNAQPGDPNAAPPPADPNAPPPPVIAPNAPQP VRIDNPVGGFSFALPAGWVESDAAHFDYGSALLSKTTGDPPFPGQPPPVANDTRIV LGRLDQKLYASAEATDSKAAARLGSDMGEFYMPYPGTRINQETVSLDANGVSGSAS YYEVKFSDPSKPNGQIWTGVIGSPAANAPDAGPPQRWFVVWLGTANNPVDKGAAKA LAESIRPLVAPPPAPAPAPAEPAPAPAPAGEVAPTPTTPTPQRTLPA (SEQ ID NO:9, TUBERCULIST No.Rv1860, on March 1st, 2007 provided, draw at this and to be reference, be also referred to as Apa, modD, mpt32)

5.MLLALLRQHIRPYRRLVAMLMMLQLVSTLASLYLPTVNAAIVDDGVAKGDTATIVRLGAVMLGVTGLQVLCAIGAVYLGSRTGAGFGRDLRSAMFEHIITFSERETARFGAPTLLTRSTNDVRQILFLVQMTATVLVTAPIMCVGGIIMAIHQEAALTWLLLVSVPILAVANYWIISHMLPLFRRMQSLIDGINRVMRDQLSGVRVVRAFTREGYERDKFAQANTALSNAALSAGNWQALMLPVTTLTINASSVALIWFGGLRIDSGQMQVGSLIAFLSYFAQILMAVLMATMTLAVLPRASVCAERITEVLSTPAALGNPDNPKFPTDGVTGVVRLAGATFTYPGADCPVLQDISLTARPGTTTAIVGSTGSGKSTLVSLICRLYDVTAGAVLVDGIDVREYHTERLWSAIGLVPQRSYLFSGTVADNLRYGGGPDQVVTEQEMWEALRVAAADGFVQTDGLQTRVAQGGVNFSGGQRQRLAIARAVIRRPAIYVFDDAFSALDVHTDAKVHASLRQVSGDATIIVVTQRISNAAQADQVIVVDNGKIVGTGTHETLLADCPTYAEFAASQSLSATVGGVG ( SEQ ID NO：10； TUBERCULIST No.Rv1273c; On March 1st, 2007 provided, and drew at this to be reference)

6.MSYVIAAPEMLATTAADVDGIGSAIRAASASAAGPTTGLLAAAADEVSSAAAAL FSEYARECQEVLKQAAAFHGEFTRALAAAGAAYAQAEASNTAAMSGTAGSSGALGS VGMLSGNPLTALMMGGTGEPILSDRVLAIIDSAYIRPIFGPNNPVAQYTPEQWWPF IGNLSLDQSIAQGVTLLNNGINAELQNGHDVVVFGYSQSAAVATNEIRALMALPPG QAPDPSRLAFTLIGNINNPNGGVLERYVGLYLPFLDMSFNGATPPDSPYQTYMYTG QYDGYAHNPQYPLNILSDLNAFMGIRWVHNAYPFTAAEVANAVPLPTSPGYTGNTH YYMFLTQDLPLLQPIRAIPFVGTPIAELIQPDLRVLVDLGYGYGYADVPTPASLFA PINPIAVASALATGTVQGPQAALVSIGLLPQSALPNTYPYLPSANPGLMFNFGQSS VTELSVLSGALGSVARLIPPIA (SEQ IDNQ:11, TUBERCULIST No.Rv0159c, on March 1st, 2007 provided, draw at this and to be reference, be also referred to as PE3 or PE).

7.MEFPVLPPEINSVLMYSGAGSSPLLAAAAAWDGLAEELGSAAVSFGQVTSGLTAGVWQGAAAAAMAAAAAPYAGWLGSVAAAAEAVAGQARVVVGVFEAALAATVDPALVAANRARLVALAVSNLLGQNTPAIAAAEAEYELMWAADVAAMAGYHSGASAAAAALPAFSPPAQALGGGVGAFLTALFASPAKALSLNAGLGNVGNYNVGLGNVGVFNLGAGNVGGQNLGFGNAGGTNVGFGNLGNGNVGFGNSGLGAGLAGLGNIGLGNAGSSNYGFANLGVGNIGFGNTGTNNVGVGLTGNHLTGIGGLNSGTGNIGLFNSGTGNVGFFNSGTGNFGVFNSGNYNTGVGNAGTASTGLFNAGNFNTGVVNVGSYNTGSFNAGDTNTGGFNPGGVNTGWLNTGNTNTGIANSGNVNTGAFISGNFNNGVLWVGDYQGLFGVSAGSSIPAIPIGLVLNGDIGPITIQPIPILPTIPLSIHQTVNLGPLVVPDIVIPAFGGGIGIPINIGPLTITPITLFAQQTFVNQLPFPTFSLGKITIPQIQTFDSNGQLVSFIGPIVIDTTIPGPTNPQIDLTIRWDTPPITLFPNGISAPDNPLGLLVSVSISNPGFTIPGFSVPAQPLPLSIDIEGQIDGFSTPPITIDRIPLTVGGGVTIGPITIQGLHIPAAPGVGNTTTAPSSGFFNSGAGGVSGFGNVGAGSSGWWNQAPSALLGAGSGVGNVGTLGSGVLNLGSGISGFYNTSVLPFGTPAAVSGIGNLGQQLSGVSAAGTTLRSMLAGNLGLANVGNFNTGFGNVGDVNLGAANIGGHNLGLGNVGDGNLGLGNIGHGNLGFANLGLTAGAAGVGNVGFGNAGINNYGLANMGVGNIGFANTGTGNIGIGLVGDHRTGIGGLNSGIGNIGLFNSGTGNVGFFNSGTGNFGIGNSGRFNTGIGNSGTASTGLFNAGSFSTGIANTGDYNTGSFNAGDTNTGGFNPGGINTGWFNTGHANTGLANAGTFGTGAFMTGDYSNGLLWRGGYEGLVGVRVGPTISQFPVTVHAIGGVGPLHVAPVPVPAVHVEITDATVGLGPFTVPPISIPSLPIASITGSVDLAANTISPIRALDPLAGSIGLFLEPFRLSDPFITIDAFQVVAGVLFLENIIVPGLTVSGQILVTPTPIPLTLNLDTTPWTLFPNGFTIPAQTPVTVGMEVANDGFTFFPGGLTFPRASAGVTGLSVGLDAFTLLPDGFTLDTVPATFDGTILIGDIPIPIIDVPAVPGFGNTTTAPSSGFFNTGGGGGSGFANVGAGTSGWWNQGHDVLAGAGSGVANAGTLSSGVLNVGSGISGWYNTSTLGAGTPAVVSGIGNLGQQLSGFLANGTVLNRSPIVNIGWADVGAFNTGLGNVGDLNWGAANIGAQNLGLGNLGSGNVGFGNIGAGNVGFANSGPAVGLAGLGNVGLSNAGSNNWGLANLGVGNIGLANTGTGNIGIGLVGDYQTGIGGLNSGSGNIGLFNSGTGNVGFFNTGTGNFGLFNSGSFNTGIGNSGTGSTGLFNAGNFNTGIANPGSYNTGSFNVGDTNTGGFNPGDINTGWFNTGIMNTGTRNTGALMSGTDSNGMLWRGDHEGLFGLSYGITIPQFPIRITTTGGIGPIVIPDTTILPPLHLQITGDADYSFTVPDIPIPAIHIGINGVVTVGFTAPEATLLSALKNNGSFISFGPITLSNIDIPPMDFTLGLPVLGPITGQLGPIHLEPIVVAGIGVPLEIEPIPLDAISLSESIPIRIPVDIPASVIDGISMSEVVPIDASVDIPAVTITGTTISAIPLGFDIRTSAGPLNIPIIDIPAAPGFGNSTQMPSSGFFNTGAGGGSGIGNLGAGVSGLLNQAGAGSLVGTLSGLGNAGTLASGVLNSGTAISGLFNVSTLDATTPAVISGFSNLGDHMSGVSIDGLIAILTFPPAESVFDQIIDAAIAELQHLDIGNALALGNVGGVNLGLANVGEFNLGAGNVGNINVGAGNLGGSNLGLGNVGTGNLGFGNIGAGNFGFGNAGLTAGAGGLGNVGLGNAGSGSWGLANVGVGNIGLANTGTGNIGIGLTGDYRTGIGGLNSGTGNLGLFNSGTGNIGFFNTGTGNFGLFNSGSYSTGVGNAGTASTGLFNAGNFNTGLANAGSYNTGSLNVGSFNTGGVNPGTVNTGWFNTGHTNTGLFNTGNVNTGAFNSGSFNNGALWTGDYHGLVGFSFSIDIAGSTLLDLNETLNLGPIHIEQIDIPGMSLFDVHEIVEIGPFTIPQVDVPAIPLEIHESIHMDPIVLVPATTIPAQTRTIPLDIPASPGSTMTLPLISMRFEGEDWILGSTAAIPNFGDPFPAPTQGITIHTGPGPGTTGELKISIPGFEIPQIATTRFLLDVNISGGLPAFTLFAGGLTIPTNAIPLTIDASGALDPITIFPGGYTIDPLPLHLALNLTVPDSSIPIIDVPPTPGFGNTTATPSSGFFNSGAGGVSGFGNVGSNLSGWWNQAASALAGSGSGVLNVGTLGSGVLNVGSGVSGIYNTSVLPLGTPAVLSGLGNVGHQLSGVSAAGTALNQIPILNIGLADVGNFNVGFGNVGDVNLGAANLGAQNLGLGNVGTGNLGFANVGHGNIGFGNSGLTAGAAGLGNTGFGNAGSANYGFANQGVRNIGLANTGTGNIGIGLVGDNLTGIGGLNSGAGNIGLFNSGTGNIGFFNSGTGNFGIGNSGSFNTGIGNSGTGSTGLFNAGSFNTGVANAGSYNTGSFNAGDTNTGGFNPGTINTGWFNTGHTNTGIANSGNVGTGAFMSGNFSNGLLWRGDHEGLFSLFYSLDVPRITIVDAHLDGGFGPVVLPPIPVPAVNAHLTGNVAMGAFTIPQIDIPALTPNITGSAAFRIVVGSVRIPPVSVIVEQIINASVGAEMRIDPFEMWTQGTNGLGITFYSFGSADGSPYATGPLVFGAGTSDGSHLTISASSGAFTTPQLETGPITLGFQVPGSVNAITLFPGGLTFPATSLLNLDVTAGAGGVDIPAITWPEIAASADGSVYVLASSIPLINIPPTPGIGNSTITPSSGFFNAGAGGGSGFGNFGAGTSGWWNQAHTALAGAGSGFANVGTLHSGVLNLGSGVSGIYNTSTLGVGTPALVSGLGNVGHQLSGLLSGGSAVNPVTVLNIGLANVGSHNAGFGNVGEVNLGAANLGAHNLGFGNIGAGNLGFGNIGHGNVGVGNSGLTAGVPGLGNVGLGNAGGNNWGLANVGVGNIGLANTGTGNIGIGLTGDYQTGIGGLNSGAGNLGLFNSGAGNVGFFNTGTGNFGLFNSGSFNTGVGNSGTGSTGLFNAGSFNTGVANAGSYNTGSFNVGDTNTGGFNPGSINTGWLNAGNANTGVANAGNVNTGAFVTGNF SNGILWRGDYQGLAGFAVGYTLPLFPAVGADVSGGIGPITVLPPIHIPPIPVGFAAVGGIGPIAIPDISVPSIHLGLDPAVHVGSITVNPITVRTPPVLVSYSQGAVTSTSGPTSEIWVKPSFFPGIRIAPSSGGGATSTQGAYFVGPISIPSGTVTFPGFTIPLDPIDIGLPVSLTIPGFTIPGGTLIPTLPLGLALSNGIPPVDIPAIVLDRILLDLHADTTIGPINVPIAGFGGAPGFGNSTTLPSSGFFNTGAGGGSGFSNTGAGMSGLLNAMSDPLLGSASGFANFGTQLSGILNRGAGISGVYNTGALGVVTAAVVSGFGNVGQQLSGLLFTGVGP ( SEQ IDNO：12； TUBERCULIST No.Rv3350c; On March 1st, 2007 provided; Draw at this and to be reference, be also referred to as PPE56 or PPE).

In other embodiments, the Mtb polypeptide comprises ESAT-6 or is made of it basically or is made of it:

MTEQQWNFAGIEAAASAIQGNVTSIHSLLDEGKQSLTKL

AAWGGGSGSEAYQGVQQKWDATATELNNALQNLARTISEAG

QAMASTEGNVTGMFA

(SEQ?ID?NO：39)

The peptide that uses:

MTEQQWNFAGIEAAA SEQ?ID?NO：40

QWNFAGIEAAASAIQ SEQ?ID?NO：41

AGIEAAASAIQGNVT SEQ?ID?NO：42

AAASAIQGNVTSIHS SEQ?ID?NO：43

AIQGNVTSIHSLLDE SEQ?ID?NO：44

NVTSIHSLLDEGKQS SEQ?ID?NO：45

IHSLLDEGKQSLTKL SEQ?ID?NO：46

LDEGKQSLTKLAAAWG SEQ?ID?NO：47

KQSLTKLAAAWGGSG SEQ?ID?NO：48

TKLAAAWGGSGSEAY SEQ?ID?NO：49

AAWGGSGSEAYQGVQ SEQ?ID?NO：50

GSGSEAYQGVQQKWD SEQ?ID?NO：51

EAYQGVQQKWDATAT SEQ?ID?NO：52

GVQQKWDATATELNN SEQ?ID?NO：53

KWDATATELNNALQN SEQ?ID?NO：54

TATELNNALQNLART SEQ?ID?NO：55

LNNALQNLARTISEA SEQ?ID?NO：56

LQNLARTISEAGQAM SEQ?ID?NO：57

ARTISEAGQAMASTE SEQ?ID?NO：58

SEAGQAMASTEGNVT SEQ?ID?NO：59

QAMASTEGNVTGMFA SEQ?ID?NO：60

In other embodiments, the Mtb polypeptide comprises CFP-10 or is made of it basically or is made of it:

MAEMKTDAATLAQEAGNFERISGDLKTQIDQVESTAGSLQGQWRG

AAGTAAQAAVVRFQEAANKQKQELDEISTNIRQAGVQYSRADEEQQ

QALSSQMG

(SEQ?ID?NO：61)

The peptide that uses:

MAEMKTDAATLAQEA SEQ?ID?NO：62

KTDAATLAQEAGNFE SEQ?ID?NO：63

ATLAQEAGNFERISG SEQ?ID?NO：64

QEAGNFERISGDLKT SEQ?ID?NO：65

NFERISGDLKTQIDQ SEQ?ID?NO：66

ISGDLKTQIDQVEST SEQ?ID?NO：67

LKTQIDQVESTAGSL SEQ?ID?NO：68

IDQVESTAGSLQGQW SEQ?ID?NO：69

ESTAGSLQGQWRGAA SEQ?ID?NO：70

GSLQGQWRGAAGTAA SEQ?ID?NO：71

GQWRGAAGTAAQAAV SEQ?ID?NO：72

GAAGTAAQAAVVRFQ SEQ?ID?NO：73

TAAQAAVVRFQEAAN SEQ?ID?NO：74

AAVVRFQEAANKQKQ SEQ?ID?NO：75

RFQEAANKQKQELDE SEQ?ID?NO：76

AANKQKQELDEISTN SEQ?ID?NO：77

QKQELDEISTNIRQA SEQ?ID?NO：78

LDEISTNIRQAGVQY SEQ?ID?NO：79

STNIRQAGVQYSRAD SEQ?ID?NO：80

RQAGVQYSRADEEQQ SEQ?ID?NO：81

VQYSRADEEQQQALS SEQ?ID?NO：82

RADEEQQQALSSQMG SEQ?ID?NO：83

Other Mtb polypeptide that use are disclosed in the U.S. Patent application No.2005/0208594 that has announced, PCT announces No.WO 2005/0909988, the U.S. Patent application No.2003/0147897 that has announced, the U.S. Patent application No.2004/01151211 that has announced, the U.S. Patent application No.2005/0272104 that has announced, the U.S. Patent application No.2006/0024332 that has announced, the U.S. Patent application No.2006/0115847 that has announced, the U.S. Patent application No.2007/0009547 that has announced, among the U.S. Patent application No.2007/0184073 that has announced, drawing in full with it at this is reference.Can use more than one Mtb polypeptide.In several embodiments, ESAT-6 (SEQ ID NO:39) and/or CFP-10 (SEQ ID NO:61) in method disclosed herein, have been used.

In another embodiment, the Mtb polypeptide that uses in method disclosed herein has and the middle amino acid sequence 75% that shows of one of SEQ ID NO:1-12 or 39-83, the sequence of 85%, 90%, 95%, 96%, 97%, 98% or 99% homology at least.For example, polypeptide can have with SEQ ID NO:1-12 or 39-83 in the amino acid sequence of one of the amino acid sequence that shows at least 85%, 90%, 95%, 96%, 97%, 98% or 99% homology.Can use the amino acid sequence of the computer program of acquisition and this paper proposition on the internet easily to obtain exemplary sequence.In an example, polypeptide has kept the function of Mtb albumen, for example combines the function of the antibodies of Mtb epi-position with specificity.

A small amount of modification of the one-level amino acid sequence of Mtb polypeptide can produce and compare the peptide with the activity that equates basically with the corresponding polypeptide of unmodified described herein.Such modification can perhaps can be spontaneous as have a mind to design by site-directed mutagenesis.This paper has comprised all polypeptide of modifying generation by these.Therefore, concrete, the non-limiting instance of Mtb polypeptide are the conservative variant of Mtb polypeptide.The form of conservative replacement is provided herein.Can be manufactured on the replacement of the amino acid sequence that shows among SEQ ID NO:1-12 and the 39-83 according to this form.In several embodiments, one at the most, two at the most, three at the most, four or five conservative replacements at the most at the most in the Mtb polypeptide, have been introduced.

The Mtb polypeptide that can be used for detecting at the immune response of Mtb is disclosed herein.These peptides comprise at least 9 amino acid or are made of it, 9 to 20 amino acid whose continuous amino acids of routine Mtb polypeptide as set forth above.Concrete non-limiting instance is 12,11,10 amino acid or 9 continuous amino acid of one of Mtb polypeptide of proposing above.In these examples, the Mtb polypeptide does not comprise the full length amino acid sequence that SEQ ID NO:1-12, SEQ ID NO:39 and/or SEQ ID NO:61 show.

Disclose the polypeptide that comprises from the separation of 9 to 12 continuous amino acids of Mtb polypeptide, wherein the polypeptide of Fen Liing comprises the amino acid sequence that is shown as QTVEDEARRMW (SEQ ID NO:13).In certain embodiments, the length of polypeptide is 9,10 or 11 amino acid.In other embodiments, polypeptide is made of the shown amino acid sequence of SEQ ID NO:13.Disclose the polypeptide that comprises from the separation of 9 to 12 continuous amino acids of Mtb polypeptide, wherein the polypeptide of Fen Liing comprises the amino acid sequence that is shown as VSAAIAGLF (SEQ ID NO:14).In certain embodiments, the length of polypeptide is 9,10 or 11 amino acid.In other embodiments, polypeptide is made of the shown amino acid sequence of SEQ ID NO:14.

In other embodiments, the length of polypeptide is 9 to 12 continuous amino acids, and comprises one of shown amino acid sequence of SEQ ID NO:40-60 or SEQ ID NO:65-83, or is made of it basically or is made of it.

In several embodiments, isolated M tb polypeptide is included in the fusion.Therefore, fusion can comprise Mtb polypeptide (referring to above) and second allos part, for example myc albumen, enzyme or the carrier that links to each other with the Mtb polypeptid covalence (for example hepatitis carrier protein or bovine serum albumin(BSA)).In several examples, 9 to 12 polypeptide in conjunction with I class MHC that amino acid constitutes by one of shown amino acid sequence of SEQ ID NO:1-14, SEQ ID NO:39 or SEQ ID NO:61 link to each other with the carrier covalency.In other examples, constitute or, link to each other with the carrier covalency by the polypeptide that one of shown amino acid sequence of SEQ ID NO:40-60 or 65-83 constitutes by one of shown amino acid sequence of one of SEQ ID NO:1-14.

In other examples, polypeptide can be a fusion, and can comprise the sequence (length that for example is not included among the SEQ ID NO:1 is at least 9 amino acid whose amino acid sequences) of allos concerning Mtb.Therefore, in several concrete limiting examples, immunogenic peptide is a fused polypeptide, and for example this polypeptide comprises 6 continuous histidine residues, beta galactosidase amino acid sequence or immunoglobulin amino acid sequence.Polypeptide also can link to each other with the carrier covalency.In other embodiments, albumen is made of the Mtb polypeptide.

Polypeptide can be chosen the repetition that comprises one or more Mtb polypeptide disclosed herein wantonly.In a concrete limiting examples, polypeptide comprises 2,3,4,5 or nearly 10 repetitions of one of above-mentioned Mtb polypeptide.Alternatively, in fused polypeptide, can comprise an above polypeptide.Therefore, in several examples, polypeptide can comprise at least two, at least three, at least four, at least five or at least six SEQ ID NO:1-14 and/or the shown amino acid sequence of SEQ ID NO:39-83.Between the Mtb polypeptide, can choose wantonly and comprise joint sequence.

Mtb polypeptide disclosed herein can be by the standard method chemosynthesis, or can recombinant production.The illustrative methods that is used for polypeptide production is described in Lu etc., and Federation of European Biochemical Societies Letters.429:31-35 is in 1998.They also can separate by the method in comprising preparative scale chromatography and immunity being separated in.

If desired, polypeptide also can pass through the emerging technology chemosynthesis.A kind of such method is described in W.Lu etc., and Federation of European Biochemical Societies Letters.429:31-35 is in 1998.Polypeptide also can use molecular genetic techniques to produce, and for example the nucleic acid by will encode Mtb or its epi-position is inserted in the expression vector, and expression vector is imported host cell, and isolated polypeptide (vide infra).

The polynucleotide of the Mtb polypeptide disclosed herein of encoding also are provided.Exemplary nucleotide sequence shows below:

ESXJ (ESAT-6 sample albumen 2)

atggcctcgcgttttatgacggatccgcacgcgatgcgggacatggcgggccgttttgaggtgcacgcccagacggtggaggacgaggctcgccggatgtgggcgtccgcgcaaaacatctcgggcgcgggctggagtggcatggccgaggcgacctcgctagacaccatgacccagatgaatcaggcgtttcgcaacatcgtgaacatgctgcacggggtgcgtgacgggctggttcgcgacgccaacaactacgaacagcaagagcaggcctcccagcagatcctcagcagctga

(SEQ?ID?NO：15)

ESXK (ESAT-6 sample albumen 3)

atggcctcacgttttatgacggatccgcacgcgatgcgggacatggcgggccgttttgaggtgcacgcccagacggtggaggacgaggctcgccggatgtgggcgtccgcgcaaaacatttccggtgcgggctggagtggcatggccgaggcgacctcgctagacaccatggcccagatgaatcaggcgtttcgcaacatcgtgaacatgctgcacggggtgcgtgacgggctggttcgcgacgccaacaactacgagcagcaagagcaggcctcccagcagatcctcagcagctaa

(SEQ?ID?NO：16)

ESXM (ESAT-6 sample albumen ESXM)

atggcctcacgttttatgacggatccgcatgcgatgcgggacatggcgggccgttttgaggtgcacgcccagacggtggaggacgaggctcgccggatgtgggcgtccgcgcaaaacatttccggtgcgggctggagtggcatggccgaggcgacctcgctagacaccatgacctagatgaatcaggcgtttcgcaacatcgtgaacatgctgcacggggtgcgtgacgggctggttcgcgacgccaacaactacgaacagcaagagcaggcctcccagcagatcctgagcagctag

(SEQ?ID?NO：17)

ESXP (ESAT-6 sample albumen 7)

atggcaacacgttttatgacggatccgcacgcgatgcgggacatggcgggccgttttgaggtgcacgcccagacggtggaggacgaggctcgccggatgtgggcgtccgcgcaaaacatctcgggcgcgggctggagtggcatggccgaggcgacctcgctagacaccatggcccagatgaatcaggcgtttcgcaacatcgtgaacatgctgcacggggtgcgtgacgggctggttcgcgacgccaacaactacgagcagcaagagcaggcctcccagcagatcctcagcagctaa

(SEQ?ID?NO：18)

ESXW (ESAT-6 sample protein 10)

atgacctcgcgttttatgacggatccgcacgcgatgcgggacatggcgggccgttttgaggtgcacgcccagacggtggaggacgaggctcgccggatgtgggcgtccgcgcaaaacatttccggcgcgggctggagtggcatggccgaggcgacctcgctagacaccatgacccagatgaatcaggcgtttcgcaacatcgtgaacatgctgcacggggtgcgtgacgggctggttcgcgacgccaacaactacgaacagcaagagcaggcctcccagcagatcctcagcagctga

(SEQ?ID?NO：19)

PE9 (PE family protein)

atgtcatacatgattgccacaccagcggcgttgacggcggcggcaacggatatcgacgggattggctcggcggttagcgttgcgaacgccgcggcggtcgccgcgacaaccggagtgctggccgccggtggcgatgaagtgttggcggccatcgctaggctgttcaacgcaaacgccgaggaatatcacgccctcagcgcgcaggtggcggcgtttcaaaccctgtttgtgcgcaccttgactggggggtgcggagtctttcgccggcgccgaggccgccaatgcgtcacagctgcagagcatcgcgcggcaggtgcggggcgccgtcaacgccgtcgccggtcaggtgacgggcaatggcggctccggcaacagcggcacttcggctgcggcggccaacccgaattccgacaacacagcGagcatcgccgatag

(SEQ?ID?NO：20)

PE PGRS42 (PE-PGRS family protein)

gtgtcgttggtgatcgcgacgccgcagctgctggcaactgcggctttggatttagcgagtattggttcgcaggtgagcgcggctaatgcggccgcggcgatgccgacgacggaagtggtggctgcggctgccgatgaagtgtcggcggcgattgcggggttgttcggggcccatgctcggcagtatcaggcgctcagcgtacaggtggcagcgtttcacgagcagtttgtgcaggcgttgactgcggccgcgggtcggtatgccagcactgaggccgctgttgagcggagtctgctgggtgcggtgaatgcgcccaccgaggcgcttttggggcgcccgttgatcggaaacggcgccgacgggacggcacccgggcagcctggcgcggccggcgggttgctgtttggcaacggtggcaacggcgcggctggcgggttcggtcaaaccggcggcagcggaggcgcggccgggttgatcggcaacggcggcaacggcggggccggtggtaccggcgcggccggcggtgccggtgggaacggggggtggttgtggggcaacggcggcaacggcggtgtcggcggcaccagcgtggccgcaggcatcgggggtgcgggcggtaacggcggcaacgccgggctgttcggccatggcggcgccggtggtaccggcggcgccggcctcgccggggcaaacggggtcaatcccacgcccggccccgcggccagcaccggggacagcccggcagatgtgtccggcatcggtgatcaaaccggcggcgacggcggcacgggcggccatggcactgccggcacgccgaccggtggcaccggcggcgacggtgccaccgcgacggcaggctcgggcaaggccaccggcggtgccggtggtgacggcggtaccgccgctgccggtggcggcggcggcaacggcggcgacggcggagtcgcgcagggcgacattgcgagcgcctttggcggtgatggtggcaacgggtccgacggtgtagccgccggcagtgggggtggtagcggcggcgccggaggcggcgctttcgtacacatcgccactgccacctctaccggtggtagcggcggtttcggtggtaacggggctgccagtgccgcctccggcgccgacggtggcgcagggggagctggcggcaatggtggcgccggcgggttgctattcggtgatggcggcaacggtggcgccggtggcgcgggtggtatcggtggtgacggcgccacgggggggcccgggggaagcggcggcaacgctggcatcgcgaggtttgacagcccagaccccgaggcagaacccgatgtggtcggcggcaagggtggtgatggcggcaagggcggcagcggccttggcgtcggcggcgccggcgggaccggcggcgcgggcggcaacggcggcgccggcgggttgttgttcggcaacggcggcaacggcggcaacgccggggccggcggggatggcggcgccggcgttgccggtggggttggcggtaacggcggcggtggtggcaccgcgacgtttcacgaagacccggtcgctggtgtctgggcggtcggtggcgtaggtggtgatggtggctccggcggcagctcgcttggtgtcggcggggtgggcggagccggtggcgtgggtggcaagggtggcgccagcggcatgttgatcggcaacggcggcaacggtggcagcggcggagtcggtggggccggtggagtcggcggggctggcggtgacggcggcaacggcggctccggtggcaacgccagtacttttggcgatgagaactccatcggcggggccggcgggacgggcggcaacgggggcaacggcgcaaacggcggtaacggtggcgctggcggtattgccggcggtgcgggtgggtccggagggttcctcagcggtgccgcaggagtcagcggcgctgacggtatcggtggcgcgggcggcgcaggcggtgccggtggcgcgggcggtagcggcggtgaggcaggcgcggggggcctcaccaacggccccgggtcccctggcgtttccggcaccgaaggcatggccggcgcgcccggctag

(SEQ?ID?NO：21)

Rv1860 (fibronectin binding protein)

atgcatcaggtggaccccaacttgacacgtcgcaagggacgattggcggcactggctatcgcggcgatggccagcgccagcctggtgaccgttgcggtgcccgcgaccgccaacgccgatccggagccagcgcccccggtacccacaacggccgcctcgccgccgtcgaccgctgcagcgccacccgcaccggcgacacctgttgcccccccaccaccggccgccgccaacacgccgaatgcccagccgggcgatcccaacgcagcacctccgccggccgacccgaacgcaccgccgccacctgtcattgccccaaacgcaccccaacctgtccggatcgacaacccggttggaggattcagcttcgcgctgcctgctggctgggtggagtctgacgccgcccacttcgactacggttcagcactcctcagcaaaaccaccggggacccgccatttcccggacagccgccgccggtggccaatgacacccgtatcgtgctcggccggctagaccaaaagctttacgccagcgccgaagccaccgactccaaggccgcggcccggttgggctcggacatgggtgagttctatatgccctacccgggcacccggatcaaccaggaaaccgtctcgctcgacgccaacggggtgtctggaagcgcgtcgtattacgaagtcaagttcagcgatccgagtaagccgaacggccagatctggacgggcgtaatcggctcgcccgcggcgaacgcaccggacgccgggccccctcagcgctggtttgtggtatggctcgggaccgccaacaacccggtggacaagggcgcggccaaggcgctggccgaatcgatccggcctttggtcgccccgccgccggcgccggcaccggctcctgcagagcccgctccggcgccggcgccggccggggaagtcgctcctaccccgacgacaccgacaccgcagCggaccttaccggcctga

(SEQ?ID?NO：22)

Rv1273c (possible medicament transport is striden film ATP in conjunction with albumin A BC transport protein)

atgctcctggccctgctgcgccagcacatccgaccgtaccgccggctggtcgcgatgctgatgatgctgcagctggtcagcaccctggcttcgctatacctcccgacggtcaacgccgcaatcgtcgacgacggcgtcgccaagggcgacaccgccaccatcgtacggctgggtgcggtgatgcttggggtgaccggattgcaggtgctgtgcgcgatcggggcggtctatctgggctcccggaccggggcgggtttcggccgtgacctgcgctcggcaatgttcgaacacatcatcaccttctcggaacgcgagaccgcccgattcggcgctccgacgttgttgacccgcagcaccaacgacgtccggcagatcctgttcctggtccagatgaccgccaccgtgctggtcaccgcaccgatcatgtgcgtcggcggaatcatcatggccatccaccaggaggccgcgctgacatggctgctgctggtcagcgttccgattctggccgtagcaaactactggatcatctcccacatgctgccgctcttccgccgcatgcagagcctgatcgacggcatcaaccgggtgatgcgcgatcagctgtccggggtgcgagtggtccgcgccttcacccgcgaaggctatgaacgcgacaagttcgcgcaggccaatacggcgctgtcgaatgccgcactgagcgccggcaactggcaagcactgatgctgccggtgaccacgctgaccatcaacgcatccagcgtcgcactgatctggttcggtgggctacgcatcgacagcggccagatgcaggtcggctccctgatcgccttcctgtcctacttcgcccagatcctgatggcggtgttgatggcgaccatgacgctggccgtgctgccacgagcgtcggtctgcgccgaacgcatcaccgaggtgctttccacgcccgccgcactcggtaaccccgacaatcccaagttcccgacggacggggtcacgggcgtagtgcgcttggctggcgcaacctttacctatcctggcgccgactgcccggtgctgcaggacatttcgttgactgcgcggcccggtaccaccaccgcgatcgtcggcagtaccggttcgggcaagtcgacactggtgtcgttgatctgccggctctacgacgtcaccgctggcgcggtcttggttgacggtatcgacgtccgcgagtaccacaccgagcggctctggtcagcgatcgggctggtgccccagcgcagctacctcttctccggaaccgtcgcggacaacctgcgctacggcgggggcccagaccaggtagtcaccgagcaggagatgtgggaggcgctgcgggtcgccgcggccgacggctttgtacaaacagacgggctgcagacgcgtgtcgcccaaggtggtgtcaacttctccggcgggcagcgccaacggctggcgatagcccgagcggtcatccgacgtccggccatctatgtgttcgacgacgcgttctccgcacttgacgtgcacaccgacgccaaagtccacgcatcgctgcgacaggtatctggtgatgcaaccatcattgttgttacacaacggatttcgaatgccgctcaggccgaccaggtcatcgttgtcgataacggtaagatcgtcggcacgggcacccacgaaacgctgctggccgattgccccacctatgccgaattcgccgcctcacaatcgctgagcgccacggtcgggggtGtagggtga

(SEQ?ID?NO：23)

Rv0159c (PE family protein)

atgtcctacgtcatcgcggccccggagatgttggcaacgacggccgcggacgtggacgggatcggttcggcgatacgagcggccagcgcgtccgctgcgggtccaacgaccggactgctggccgcggccgccgatgaggtgtcgtcggccgctgcagcgctgttcagcgaatacgcgcgcgaatgtcaagaggtcctaaagcaggctgcggcgttccatggcgagttcacccgggcgctggctgccgccggggccgcctatgcccaggctgaagccagcaacaccgctgctatgtcgggcaccgccgggtccagcggcgccctcggttctgtcgggatgctgtcaggcaacccgctaaccgcgttgatgatgggcggcaccggggaaccgatccttagtgaccgcgtcttggcgatcattgacagcgcatacattcggcccattttcgggcccaacaacccggtcgcccagtacacgcccgagcagtggtggccgtttatcgggaacctgtcactggaccaatccatcgcccagggtgtcacgctgctgaacaacggcatcaacgcggaactacaaaatgggcatgacgtcgtcgttttcggctactcgcaaagcgccgcggtagcgaccaatgaaatacgcgctcttatggcgttaccaccgggccaagccccagatccaagccggctggctttcacgttgatcggtaatatcaataaccccaacggcggcgtcctcgagcgttacgtgggcctttacctcccgttcttggatatgtcgttcaacggtgcgactccaccggattccccctaccagacctacatgtacaccggccaatacgacggctacgcccacaacccgcagtacccgctcaatatcttgtcggacctcaacgccttcatgggcatcagatgggtgcacaacgcgtaccccttcaccgcggccgaggttgccaatgccgtgccgttgcccacgtctccgggctacaccggcaacacccattactacatgtttctgacccaggacctgccgctgttgcagccgattcgcgccatccccttcgtagggaccccaatagccgagctgattcagcccgacctacgggtgctagtcgacttgggctatggctacggctacgccgacgtacccaccccggccagcctgttcgcgccaatcaacccgatcgccgtggcctcggccctggcgaccgggaccgtgcaaggcccccaagccgccctagtaagcatcggattgttaccgcagtccgcgctacccaatacgtatccgtatcttccgtcggcgaatccgggcctgatgttcaacttcggtcaatccagtgtgacggagttgtcggtgctcagtggcgccctcgggtccgtagcgagattgattccaccgatcgcgtga

(SEQ?ID?NO：24)

Rv3350c (PPE family protein)

atggagtttccggtgttgccaccggaaatcaactccgtgctgatgtattcgggtgcggggtcgagcccgttgctggcggcggccgcggcgtgggatgggctggctgaggagttggggtcggcggcggtgtcgtttgggcaggtgacgtcgggcctgacggcgggggtgtggcagggtgcggcggcggcggcgatggcggccgcggcggcgccgtatgcggggtggttgggttcggtggcggccgcggccgaggcggtggccgggcaggcgcgggtggtggtgggggtctttgaggcggcgttggcggcgacggtggatccggcgctggtggcggccaaccgggcgcggctggtggcgttggcggtgtcgaatctgttggggcagaacacgccggcgatcgcggccgccgaggccgagtacgagctgatgtgggccgccgatgtggcggcgatggccggctaccattccggcgcgtcggctgctgccgcggcgttgccggcgttcagcccaccggcgcaggcgctggggggaggtgtcggcgcgttccttaccgccctgttcgccagccctgcgaaggcgctgagcctgaatgcgggtttgggcaatgtcggcaattacaacgtcgggttgggcaatgtcggggtgttcaacctgggcgcgggcaatgtgggtgggcagaatctgggtttcgggaatgccggtggcaccaatgtcgggttcggcaacctcggtaacgggaatgtcgggttcggcaactccggtctgggggcgggcctggccggcttgggcaatatcgggttgggcaatgcgggcagcagcaactatggtttcgcaaacctgggtgtgggcaacatcggtttcggcaacaccggcaccaacaacgtcggcgtcgggctcaccggcaaccacctgacgggtatcgggggcctgaattcgggcaccgggaatatcgggttgttcaactccggcaccgggaatgtggggttcttcaattcggggaccgggaacttcggggtgttcaactcgggtaattacaacaccggtgtcggtaatgcggggacggccagcacggggttgttcaatgccggcaatttcaacaccggcgtggtgaacgtgggcagttacaacaccggcagtttcaacgccggcgacaccaacaccggtggcttcaaccccggcggtgtgaacaccggctggctgaacaccggcaacaccaacaccggcatcgccaactcgggcaacgtcaacaccggcgcgttcatctcgggcaacttcaacaacggcgtgctgtgggtgggtgactaccagggcctgttcggcgtctccgccggctcgtcgatccccgcaattcccatcggcctggtgctcaacggcgacatcggcccgatcaccatccagcccatcccgatcctgcccaccatcccgctcagcattcaccaaaccgtcaacttgggcccgctggtggttcccgacatcgtgatccccgccttcggcggcggtatcggcatacccatcaacatcggcccgctgaccatcacacccatcaccctgtttgcccaacagacatttgtcaaccaattgccctttcccaccttcagtttagggaaaatcacaattccacaaatccaaacctttgattctaacggtcagcttgtcagctttatcggccctatcgttatcgacaccaccattcccggacccaccaatccacagattgatttaacgatcagatgggatacccctccgatcacgctgttcccgaatggcatcagtgctcccgataatcctttggggttgctggtgagtgtgtcgatcagtaacccgggctttaccatcccgggatttagtgttcccgcgcagccgttgccgttgtcgatcgatatcgagggccagatcgacgggttcagcaccccgccgatcacgatcgatcgcatccccctgaccgtggggggcggggtcacgatcggccccatcacgatccagggccttcatatcccggcggcgccgggagtggggaacaccaccacggccccgtcgtcgggattcttcaactccggtgcgggtggggtgtcgggtttcggcaacgtcggcgcgggcagctcgggctggtggaaccaggcgccgagcgcgctgttgggggccggttcgggtgttggcaacgtgggcaccctgggctcgggtgtgctcaacctgggctcagggatctcggggttctacaacaccagcgtgttgcctttcgggacaccggcggcggtgtcgggcatcggcaacctgggccagcagctgtcgggggtgtcggcggcgggaaccacgctgcgctcgatgctcgccggcaacctcgggttggccaatgtgggcaacttcaacaccgggttcggaaatgtcggggacgtcaacctgggtgcggccaacatcggtgggcacaacctgggcctgggcaatgtcggggacggcaacctggggttgggcaacatcggccatggcaacctggggtttgccaacttgggcctgaccgccggcgcggcgggggtgggcaatgttggttttggcaatgccggcatcaacaactatggcttggcgaacatgggtgtgggcaatattgggtttgccaacaccggcacgggcaacatcgggatcgggctggtcggggaccatcggaccgggatcgggggcttgaactccggcatcggcaatatcgggttgttcaactccggcaccggcaacgtcgggttcttcaattccgggaccggcaacttcggcatcgggaactccggccgcttcaacaccgggatcggtaatagcggaacggccagcaccgggctcttcaatgccggcagcttcagcaccggcatcgccaacactggtgactacaacacgggcagcttcaacgccggcgacaccaacaccggtggcttcaacccgggcggcatcaacaccggctggttcaacaccgggcatgccaacaccgggttggccaacgcgggcaccttcggcaccggcgccttcatgacgggcgactacagcaacggcctgttgtggcggggcggctacgagggcctggtcggcgtccgcgtcgggcccacgatctcccaattcccggtcaccgtgcacgcgatcggcggggtgggcccgctgcatgtggcgcccgtcccggtacccgccgtgcacgtcgagatcaccgacgccaccgtcggcctgggtccgttcaccgtcccaccgatcagcattccctcacttcccatcgccagcatcaccggaagcgtggacctggccgcaaacaccatctcgccgattcgcgctcttgacccgctcgccggttcgatagggctttttctcgagccgttccgcctcagtgacccatttatcaccattgatgcgttccaagttgttgccggtgtcttgttcctagagaacatcattgtgcccggcctcacggttagcggtcagatattggtcaccccgacaccaattcccctaaccctcaacttggacaccaccccgtggacgcttttcccgaatggtttcaccattcccgcgcaaacccccgtgacggtgggtatggaggtcgccaacgacgggttcaccttcttcccgggtgggctgacctttccgcgggcctccgccggggtcaccggactgtccgtggggctggacgcgttcacgctgttgcccgacgggttcaccctcgacaccgtgccggcgaccttcgacggcaccatcctcatcggcgatatcccgatcccgatcatcgatgtgccggcggtgccggggttcggcaacaccaccacggccccatcgtcggggttcttcaacaccggcggcggcggtggatcggggttcgccaacgtcggcgcgggcacgtcgggctggtggaaccaggggcacgacgtgttagcaggggcgggctcgggagttgccaatgccggcacgctgagctcgggcgtgctgaacgtcggctcggggatctccgggtggtacaacaccagcaccctgggagcgggcaccccggcggtggtctcgggcatcggcaacctcggccagcagctgtcggggttcttggcaaatgggaccgtgctcaaccggagccccattgtcaatatcgggtgggccgatgtgggcgcgttcaacaccgggttgggcaatgtgggggacctcaactggggtgcggccaacatcggcgcgcagaacctgggcctgggcaatctcggcagcgggaacgtcgggttcggcaacatcggtgccggcaacgtcgggttcgccaactcgggtccggcggtgggcctggccggcctgggcaacgtggggttgagcaatgccggcagcaacaactgggggctggccaacctgggtgtgggcaacatcgggttggccaacaccggcacgggcaacatcgggatcgggctggtcggcgactaccagaccggcatcggcggcctcaactcgggtagtggcaatatcggattgttcaattccggcaccggcaatgtcgggttcttcaacaccggcaccggcaacttcggactgttcaactccggtagtttcaacaccggcatcggtaatagcggaaccggcagtactgggctcttcaatgccggcaatttcaacaccggcatcgccaaccccgggtcgtacaacacgggcagcttcaatgtcggtgataccaacaccggtggtttcaacccgggcgacatcaacaccggctggttcaacaccggcattatgaatacgggcacccgcaacaccggcgccctcatgtcggggaccgacagcaacggcatgctgtggcgcggcgaccacgagggcctgttcggcctgtcctatggcatcacgatcccgcaattcccgatccgcatcaccacgactggcggtatcggccccatcgtcatcccggacaccacgatccttccgccgctgcacctgcagatcaccggcgacgcggactacagcttcaccgtgcccgacatccccatccccgccatccacatcggcatcaatggcgtcgtcaccgtcggcttcaccgccccggaagccaccctgctgtccgccctgaagaataacggtagcttcatcagcttcggccccatcacgctctcgaatatcgatattccgcccatggatttcacgttaggcctgcccgttcttggtcctatcacgggccaactcggaccaattcatcttgagccaatcgtggtggccgggatcggtgtgcccctggagatcgagcccatccccctggatgcgatttcgttgagtgagtcgattcctatccgcatacctgttgatattccggcctcggtcatcgatgggatttcaatgtcggaagtggtgccgatcgatgcgtccgtggacatcccggcggtcacgatcacaggcaccaccatttccgcgatcccgctgggcttcgacattcgcaccagtgccggacccctcaacatcccgatcatcgacatcccggcggcgccgggcttcgggaactcgacccagatgccgtcgtcggggttcttcaacaccggtgccggcggcggatcgggcatcggcaacttgggtgcgggcgtgtcgggcctgctcaaccaggccggcgcggggtcactggtggggacactctcggggctgggcaatgccggcaccctggcctcgggtgtgctgaactccggcaccgccatctccgggctgttcaacgtgagcacgctggacgccaccaccccggcggtgatctcggggttcagcaacctcggcgaccatatgtcgggggtgtccatcgatggcctgatcgcgatcctcaccttcccacctgccgagtccgtgttcgatcagatcatcgacgcggccatcgccgagctgcagcacctcgacatcggcaacgctttggccttgggcaatgtcggcggggtgaacctcggtttggctaacgtcggtgagttcaacctgggtgcgggcaacgtcggcaacatcaacgtcggcgccggcaacctcggcggcagcaacttggggttgggcaacgtcgggaccggcaacctcgggttcggcaacatcggtgccggcaatttcggattcggcaacgcgggcctgaccgcgggcgcggggggcctgggcaatgtggggttgggtaacgccggcagcggcagctgggggttggccaacgtgggtgtgggcaatatcgggttggccaacaccggcaccggcaacatcgggatcgggctgaccggggactatcggaccgggatcggcggcctgaactcgggcaccgggaacctcgggttgttcaactcgggcaccggcaacatcgggttcttcaacaccgggaccgggaacttcgggctgttcaactcgggcagttacagcaccggtgtggggaatgcgggcacggccagcaccgggttgttcaacgcggggaacttcaacaccggtctggccaatgccggctcctacaacaccggcagcctcaacgtgggcagcttcaacaccggcggcgtcaacccgggcaccgtcaacaccggctggttcaacaccggccacaccaacaccggcctgttcaacaccggcaacgtcaacaccggcgcgttcaactccggcagcttcaacaacggggcgctgtggaccggtgactaccacgggctggtcggcttctccttcagcatcgacatcgccggcagcaccctgctggacctcaacgaaaccctcaacctgggccccatccacatcgagcagatcgacatccccggcatgtcgctgttcgacgtccacgaaatcgtcgagatcggacccttcaccatcccgcaggtcgatgttcccgcgataccgctagagatccacgaatcgatccacatggatcccatcgtcctggtgcccgccaccacaattcccgcacagacgagaaccattccgctggacatccccgcctcacccgggtcaaccatgacgcttccgctcatcagcatgcgcttcgaaggcgaggactggatcctcgggtcgaccgcggcgattcccaatttcggagaccccttcccggcgcccacccagggcatcaccattcacaccggccctggccccggaacgaccggcgagctcaagatatctattccgggtttcgagattccgcaaatcgctaccacgagattcctgttggacgtgaacatcagcggtggtctgccggccttcaccttgttcgcgggtggcctgacgatccccacgaacgccatcccgttaacgatcgatgcgtccggcgcgctggatccgatcacgattttcccgggtgggtacacgatcgacccgctgccgctgcacctggcgctgaatctcaccgtgcccgacagcagcatcccgatcatcgatgtcccgccgacgccagggttcggcaacaccacggcgaccccgtcgtcggggttcttcaactccggcgccggtggggtgtcggggttcggaaacgtcgggtcgaacctgtcgggctggtggaaccaggcggcgagcgcgctggcggggtcgggatcgggggtgttgaatgtcggcacgctgggctcgggtgtgctcaacgtcggctcgggtgtctcggggatctacaacaccagcgtgttgccgctcgggacgccggcggtgctgtcgggcctcggcaacgtcggccatcagctgtcgggcgtgtctgcggccgggaccgcgttgaaccagatccccatcctcaacatcgggttggcggatgtgggcaacttcaacgtcgggttcggcaacgtcggggacgttaacctgggcgcggccaacctcggtgcgcaaaacctggggctgggcaacgtcggcaccggcaacctcggcttcgccaacgtcggccacggcaatatcggtttcggcaattcgggtctgaccgccggcgcggccggcctgggcaacacggggttcggcaatgccggcagcgccaactatggtttcgccaaccagggcgtgcgcaacatcgggttggccaacaccggcaccggcaacatcgggatcgggctggtgggggacaacctcaccggcatcgggggcctgaactccggtgccggcaatatcggcttgttcaactccggcaccggcaacatcgggttcttcaactccgggaccggcaacttcggcatcggtaactcgggcagcttcaacaccggcatcggcaatagcggaacgggcagcactgggctcttcaatgccggcagcttcaacaccggcgtggccaacgccggcagctacaacaccggcagcttcaatgccggcgacaccaacaccggggggttcaacccgggcaccatcaacaccggctggttcaacaccggccacaccaataccggcatcgccaactcgggcaacgtcggcaccggcgcgttcatgtcgggcaacttcagcaacggcctgttgtggcggggtgatcacgagggcctgttcagcctgttctacagcctcgacgtgccccggatcaccatcgtggacgcccacctcgacggcggcttcggacccgtggtcctcccgcccatcccggtgccggccgttaatgcgcacctgaccggaaacgtcgcgatgggcgcattcaccattccgcagatcgacatccccgcactcaccccaaacatcaccggaagcgccgccttccgcatcgttgtggggtccgtgcgcattccgccggtgagtgtcattgtggagcaaataatcaacgcctcggttggggcggagatgaggatagatcccttcgaaatgtggactcaaggcactaatggccttggtataaccttctattcattcggatcggccgacggttcgccctacgccaccggcccactcgttttcggcgccggcacgagcgacggaagccatctcaccatttccgcgtccagcggggcgtttaccactccgcagctcgaaactggcccgatcacgttgggcttccaggtgcccggcagcgtcaacgcgatcaccctcttccccggtggtttgacgttcccggcgacctcgctgctgaacctggacgtgaccgccggcgccggcggcgtggacatcccggccatcacctggcccgagatcgcggcgagcgccgacggctcggtgtatgtcctcgccagcagcatcccgctgatcaacatcccgcccaccccgggcattgggaacagcaccatcaccccgtcgtcgggcttcttcaacgccggcgcgggcgggggatcgggcttcggcaacttcggcgcgggcacctcgggctggtggaaccaggcgcacaccgcgctggcgggggcgggctcgggttttgccaacgttggcacgctgcattccggtgtgctcaacctgggctcgggtgtctcggggatctacaacaccagcacgctgggggtggggaccccggcgctggtctcaggcctgggcaacgtcggccaccaactgtcggggctgctttccggcgggtccgcggtgaacccggtgaccgttctgaatatcgggttggccaacgtcggcagccacaacgccggtttcggcaatgtcggggaggtcaacctgggcgcggccaacctcggcgcgcacaacctgggcttcggaaatatcggcgccggcaacctggggttcggcaatattggccacggcaatgtcggagtcggcaactcgggtctgaccgcgggcgtgccgggcctgggcaatgtggggttgggcaatgccggcggcaacaactgggggttggccaacgtgggcgtgggcaatatcgggttggccaacaccggcaccggcaacattgggatcgggctgaccggcgactaccagaccggcatcggcggcctaaattccggtgccggcaacctggggttgttcaactccggcgccggcaacgtcgggttcttcaacaccgggaccggcaacttcgggttgttcaactccggcagcttcaacaccggcgtcggcaatagcggaacgggcagcactgggctcttcaatgccggcagtttcaacaccggtgtggccaacgccggcagctacaacacgggcagcttcaatgtcggtgacaccaacaccgggggcttcaacccgggcagcatcaacaccggctggctcaacgccggcaacgccaacaccggggtggccaacgcgggcaatgtcaacaccggcgccttcgtcaccggcaacttcagcaacggcatcctgtggcgcggcgactaccagggcctggccggcttcgccgtgggctacaccctcccgctgttccccgcggtgggcgccgacgtcagcggcgggatcggcccgattaccgtgctgccgcccatccacatcccgcccattccggtcggcttcgccgcggtcggtggcatcggcccgatcgccatcccggacatctctgttccatccattcacttgggcctcgaccccgccgtccatgtcggctccatcaccgtcaaccccattaccgtcaggaccccgcccgtgctcgtcagttactcccaaggagccgtcaccagcacgtccggaccaacctcagagatttgggtcaagcccagcttcttccccggaatccggatcgcgccctctagcggcgggggtgcaacgtccacgcaaggggcatactttgtggggcccatctccatcccctccggcacggtgaccttcccgggattcaccatccccctcgacccgatcgacatcggcctgccggtgtcgctgaccatcccggggttcaccatcccgggcggcaccctgatccccaccctcccgctgggcctcgcgttgtccaatggcatcccgcccgtcgacatcccggccatcgttctcgaccggatcttgctggacctgcacgccgacaccactatcggcccgatcaacgtcccgatcgccgggttcggcggggcgccgggtttcgggaactcgaccacgctgccgtcgtcgggcttcttcaacaccggagctggcggcggttcgggctttagcaacaccggcgcgggcatgtcgggattgctcaacgcgatgtcggatccgctgctcgggtcggcgtcgggcttcgccaacttcggcacccagctctccggcatcctcaaccgcggcgccggcatctcgggcgtgtacaacaccggcgcgctgggtgttgtcaccgcggccgtcgtctcgggtttcggcaacgtcggccagcaactgtcgggcttgctcttcaccggcgtcgggccctaa

(SEQ?ID?NO：25)

These polynucleotide comprise DNA, cDNA and the RNA sequence of the target polypeptides of encoding.Silent mutation in the coded sequence stems from the degeneracy (being redundancy) of genetic code, thus an above codon same amino acid residue of can encoding.Therefore, for example leucine can be by CTT, CTC, CTA, CTG, TTA or TTG coding; Serine can be by TCT, TCC, TCA, TCG, AGT or AGC coding; Asparagine can be by AAT or AAC coding; Aspartic acid can be by GAT or GAC coding; Halfcystine can be by TGT or TGC coding; Alanine can be by GCT, GCC, GCA or GCG coding; Glutamine can be by CAA or CAG coding; Tyrosine can be by TAT or TAC coding; Isoleucine can be by ATT, ATC or ATA coding.The form of display standard genetic code be found in various sources (L.Stryer for example, 1988, " biological chemistry " (third edition) (Biochemistry, 3rdEdition), W.H.5 Freeman and Co., NY).

The nucleic acid of coding Mtb polypeptide can be by in-vitro method clone or amplification, for example polymerase chain reaction (PCR), ligase chain reaction (LCR), based on the amplification system of transcribing (TAS), keep sequence replicating system (3SR) and Q β replicase amplification system (QB) automatically.For example, the polynucleotide of encoding proteins can use the primer based on the dna sequence dna of molecule, and the polymerase chain reaction by cDNA separates.Diversified clone and amplification in vitro method are known for the professional in present technique field.PCR method is described in for example U.S. Patent No. 4,683,195; Mullis etc., Cold Spring Harbor Symp.Quant.Biol.51:263,1987; With " round pcr " (PCR Technology) of Erlich chief editor, Stockton Press, NY, in 1989.Also can be selected from the probe of the sequence of required polynucleotide by use, polynucleotide are separated in screening-gene group library or cDNA library under tight hybridization conditions.

The polynucleotide of coding Mtb polypeptide comprise the recombinant DNA that merges in the carrier, exists in self-replicating type plasmid or the virus or in prokaryotes or the Eukaryotic genomic DNA or as the independent molecule (for example cDNA) that does not rely on other sequences.Nucleotide of the present invention can be the modified forms of ribonucleotide, deoxyribonucleotide or any nucleotide.Term comprises the DNA of strand and double chain form.

In one embodiment, used and be used at yeast saccharomyces cerevisiae (S.cerevisiae) or Kluyveromyces lactis (Kluyveromyces lactis) carrier of expressing for example.Known have several promoters to can be used in the yeast expression system, for example constitutive promoter cytoplasma membrane H ⁺-ATPase (PMA1), glyceraldehyde-3-phosphate dehydrogenase (GPD), phosphoglyceric kinase-1 (PGK1), alcohol dehydrogenase-1 (ADH1) and multidirectional drug resistance pump (PDR5).In addition, can use inducible promoter, for example the heat shock HSE element (being elevated to 37 ℃ by temperature induces) of GAL1-10 (inducing), PHO5 (inducing) and series connection by the low inorganic phosphate in extracellular by galactose.Titratable derivant is made response instructs variable expression promoter to comprise methionine response MET3 and MET25 promoter and copper dependence CUP1 promoter.Any of these promoter can be cloned in multicopy (2 μ) or single copy (CEN) plasmid, expression is provided the control of extra level.Plasmid can comprise the antibiotic resistance (AMP) that is used for the nutrition mark (for example URA3, ADE3, HIS1 etc.) that screens at yeast and is used for breeding on bacterium.It is known being used for going up the plasmid of expressing at Kluyveromyces lactis (K.lactis), for example pKLAC1.Therefore, in an example, in bacterium, after the amplification, can plasmid be imported in the corresponding yeast nutrition deficiency by transforming similar method with bacterium.

The Mtb polypeptide can be expressed in the various yeast strains.For example, in yeast host cell, deleted 7 multidirectional drug resistance transport protein YOR1, SNQ2, PDR5, YCF1, PDR10, PDR11 and PDR15 and their transcriptional factors PDR1 and PDR3 simultaneously, made the bacterial strain that obtains medicaments insensitive.The yeast strain that also can utilize plasma membrane lipid form to change is for example at the erg6 mutant strain of ergosterol biosynthesizing defective.Can in the yeast of the main vacuole endopeptidase Pep4 shortage that other vacuole hydrolytic enzymes of control activate, express the extremely sensitive albumen of proteolysis.If corresponding null mutant can not be survived, can utilize the heterogenous expression in having the allelic bacterial strain of the temperature sensitivity of gene (ts).

The viral vectors that also can prepare the Mtb polypeptide disclosed herein of encoding.Made up many viral vectors, comprise polyomavirus, SV40 (Madzak etc., 1992, J.Gen.Virol., 73:15331536), adenovirus (Berkner, 1992, Cur.Top.Microbiol.Immunol., 158:39-6; Berliner etc., 1988, Bio Techniques, 6:616-629; Gorziglia etc., 1992, J.Virol., 66:4407-4412; Quantin etc., 1992, Proc.Nad.Acad.Sci.USA, 89:2581-2584; Rosenfeld etc., 1992, Cell, 68:143-155; Wilkinson etc., 1992, Nucl.Acids Res., 20:2233-2239; Stratford-Perricaudet etc., 1990, Hum.Gene Ther., 1:241-256), vaccinia virus (Mackett etc., 1992, Biotechnology, 24:495-499), adeno-associated virus (Muzyczka, 1992, Curr.Top.Microbiol.Immunol., 158:91-123; On etc., 1990, Gene, 89:279-282), herpesviral comprises HSV and EBV (Margolskee, 1992, Curr.Top.Microbiol.Immunol., 158:67-90; Johnson etc., 1992, J.Virol., 66:29522965; Fink etc., 1992, Hum.Gene Ther.3:11-19; Breakfield etc., 1987, Mol.Neurobiol., 1:337-371; Fresse etc., 1990, Biochem.Pharmacol., 40:2189-2199), sindbis virus (H.Herweijer etc., 1995, Human Gene Therapy 6:1161-1167; U.S. Patent No. 5,091,309 and U.S. Patent No. 5,2217,879), Alphavirus (S.Schlesinger, 1993, Trends Biotechnol.11:18-22; I.Frolov etc., 1996, Proc.Natl.Acad.Sci.USA 93:11371-11377) and retroviruse (Brandyopadhyay etc., 1984, Mol.Cell Biol., the 4:749-754 of birds; Petropouplos etc., 1992, J.Virol., 66:3391-3397), retroviruse (Miller, 1992, Curr.Top.Microbiol.Immunol., the 158:1-24 of muroid; Miller etc., 1985, Mol.Cell Biol., 5:431-437; Sorge etc., 1984, Mol.Cell Biol., 4:1730-1737; Mann etc., 1985, J.Virol., 54:401-407) and the retroviruse of human origin (Page etc., 1990, J.Virol., 64:5370-5276; Buchschalcher etc., 1992, J.Virol., 66:2731-2739).Baculoviral (autographa california multinuclear polyhedrosis virus; AcMNPV) carrier also is being known in the art, and can obtain (PharMingen for example, San Diego, Calif. from commercial source; Protein Sciences Corp., Meriden, Conn.; Stratagene, La Jolla, Calif.).

Viral vectors, for example poxvirus vector of coding Mtb polypeptide comprise the expression control element that at least one and the nucleotide sequence of coding Mtb polypeptide can be operatively connected.Expressing control element is inserted in the viral vectors to control and to regulate the expression of nucleotide sequence.The example of the expression control element that uses in these carriers includes but not limited to operator and promoter region, the Yeast promoter of lac system, bacteriophage lambda and is derived from the promoter of polyomavirus, adenovirus, retroviruse or SV40.Other operating elements include but not limited to targeting sequencing, terminator codon, poly-adenosine signal and for the nucleotide sequence of coding Mtb polypeptide suitably transcribing and subsequently necessary any other sequence of translation in host system.Expression vector can comprise for the expression vector that contains nucleotide sequence in host system transfer and subsequently duplicate necessary other elements.The example of such element includes but not limited to replication origin and selective key thing.The professional in present technique field should also be appreciated that, such carrier can use conventional method easily to make up (Ausubel etc., (1987) " molecular biology modernism " (Current Protocols in Molecular Biology), John Wiley and Sons, New York N.Y) also can be purchased.

The dna sequence dna of coding Mtb polypeptide can carry out vivoexpression by DNA is transferred in the suitable host cell.Cell can be protokaryon or eukaryotic.Term also comprises the spawn of object host cell.Should be appreciated that because may undergo mutation, all offsprings can be not consistent with parental cell between replicative phase.The method of stable transfer the---mean that foreign DNA keep continuously in the host---is being known in the art.

As what point out above, the polynucleotide sequence of coding Mtb can be operatively connected with expression control sequenc.The expression control sequenc that can be operatively connected with coded sequence in the connection is so that realize the expression of coded sequence under the condition compatible with expression control sequenc.Keeping allowing the correct translation of mRNA of the proper reading frame of the initiation codon (being ATG) of the promoter that expression control sequenc includes but not limited to be fit to, enhancer, transcription terminator, protein coding gene front, the splicing signal of introne, this gene, and terminator codon.

Host cell can comprise microorganism, yeast, insect and mammalian host cell.Expressing the method for the dna sequence dna with eucaryon or virus sequence in prokaryotes, is being known in the art.The limiting examples of the host cell that is fit to comprises bacterium, ancient bacterium, insect, fungi (for example yeast), mycobacterium (for example mycobacterium smegmatis (M.smegmatis)), plant and animal cell (for example mammalian cell is for example human).The exemplary cells of using comprises the mammal myeloma and the lymphoid cell line of colon bacillus (Escherichia coli), bacillus subtilis (Bacillus subtilis), saccharomyces cerevisiae (Saccharomyces cerevisiae), salmonella typhimurium (Salmonella typhimurium), SF9 cell, C129 cell, 293 cells, Neurospora (Neurospora) and immortalization.The technology that is used at culture breeding mammalian cell is well-known (referring to Jakoby and Pastan chief editor, 1979, " cellular incubation " (Cell Culture), (Methods in Enzymology in " Enzymology method " of the 58th volume, volume 58), Academic Press, Inc., Harcourt Brace Jovanovich, N.Y.).The example of mammalian host cell line commonly used is VERO and HeLa cell, Chinese hamster ovary celI and WI38, BHK and COS clone, but also can use other clones, for example be designed to provide the cell of the required glycosylation pattern of high expressed more or other characteristics.As discussed above, the technology that is used for transformed yeast cell, for example polyglycol conversion, protoplast transformation and particle gun also are being known (referring to Gietz and Woods in the art, Methods in Enzymology 350:87-96,2002).

The routine techniques that uses the recombinant DNA transformed host cell to know by the professional in present technique field carries out.When the host is prokaryotes, during such as but not limited to Escherichia coli, can uses program well-known in the art, pass through CaCl from exponential growth after date results and subsequently ₂The cell preparation that method is handled can be taken in the competent cell of DNA.Alternatively, can use MgCl ₂Or RbCl.If desired, transform also and can behind the bioplast that forms host cell or by electroporation, be undertaken.

When the host is eucaryote, can use the DNA transfection method for example coprecipitation of calcium phosphate, the conventional mechanical program plasmid or the viral vectors that for example will be wrapped in the liposome carry out microinjection, electroporation, insertion.Eukaryotic also can with second kind of exogenous DNA molecule of the polynucleotide sequence of coding Mtb polypeptide and coding selectivity phenotype for example herpes simplex virus thymidine kinase gene carry out cotransfection.Another kind method be to use the eucaryon viral vectors for example simian virus 40 (SV40) or the instantaneous infection of bovine papilloma virus or transformed eukaryotic nucleus and expressing protein (referring to for example " eucaryon viral vectors " (Eukaryotic Viral Vectors), Cold Spring Harbor Laboratory, the Gluzman chief editor, 1982).

There are various other analytical plans that are suitable for polypeptide of the present disclosure.Above-described purpose only is exemplary.

By following non-restrictive example the disclosure is illustrated.

Embodiment

For many infection, complete CD8 reply show as that antigen enters MHC-I processing approach, peptide and/or non-peptide class antigen combines with the MHC-I molecule and these structures by the T cell recognition.Finally, relatively limited pathogen specific T cell subsets appears.Although many generally acknowledged CD4Mtb antigens (Reed etc., Microbes Infect 7:922-931,2005) (ESAT-6, CFP10, Ag85 etc.) on the books are about by human CD8 ⁺The understanding of the common Mtb antigen of T cell recognition is shockingly few.The certified CD8 epi-position of major part is definite (referring to for example Lalvani by the selected Mtb polypeptide that combines with Ia class MHC molecule (in most of the cases being HLA-A2) high-affinity being tested come, Microbes Infect 7:922-931,1998).But in nearly all these situations, the stripped frequency of these T cells in the Mtb infected individuals is low or can not be detected, shows that on behalf of immunodominance, these specificitys can not reply.On the contrary, in the limited case of the epi-position of using the T cell to determine to comprise in the selected Mtb antigen, verified high stripped frequency is (referring to Lewinsohn etc., Am J Respir Crit Care Med 166:843-848,2002), show to be that the method at center can be identified the immunodominance epi-position with the T cell.In addition, in the people that Mtb infects, detected at some and represented the cd8 t cell of the Mtb antigen of good T4 antigen (CFP10, ESAT-6, Ag85 and Mtb39) to reply with high-frequency.Therefore, use the limited library of the over lapping synt hetic peptides of several known CD4Mtb antigens of representative, determine the people who suffers from the sick infection of active tuberculosis (TB) and latent tuberculosis (LTBI) and do not infect in the object value that the CD8 at these antigens replys.In addition, use one group of Mtb specific C D8 ⁺The T cell clone is determined the minimum epi-position that is identified in these antigens, and determines the contribution that these new epi-positions are replied the Mtb specific C D8 that exsomatizes.

Embodiment 1

Materials and methods

Human subjects.Infected individuals is not defined as negative and do not have a healthy individual of the known risk factors that Mtb infects in conjunction with rhzomorph skin test (TST).But the LTBI individuality is defined as having positive TST the healthy people who does not have the sings and symptoms of activity TB.In all activity TB cases, lung TB is diagnosed by prefecture TB control center (TB Controller of the county), and confirms by the sputum Much's bacillus cultivation positive.From the separation of whole blood peripheral blood lymphocytes (PBMC) that becomes point-score to obtain by venipuncture or single blood sampling.

Nutrient culture media and reagent.Nutrient culture media is by being added with 10% hyclone (FBS; Bio Whittaker), 5X 10 ^-5The RPMI 1640 of M 2ME (Sigma-Aldrich) and 2mM glutamine (GIBCOBRL) constitutes.For Mtb reaction-ive T cell clone's growth and analysis, in RPMI 1640, augmented 10% human serum.(American Type Culture Collection (Rockville, MD)) obtains Mtb bacterial strain H37Rv, and prepares (Lewinsohn etc., J Immunol165:925-930,2000) according to former description from American type culture collection.Peptide is by Genemed Synthesis, and (San Francisco CA) synthesizes in Inc company.The synthetic peptide merging thing that 15 overlapping aggressiveness of 11 amino acid (aa) constitute that has by representing Mtb albumen is proved to be strong T4 antigen.Representative CFP-10 (Berthet etc., Microbiology 144:3195-3203,1998 have been synthesized; Dillon etc., J Clin Microbiol 38:3285-3290,2000), (two kinds merge things, A ﹠amp for ESAT-6 (Sorenson etc., Infect Immun63:1710-1717,1995), Mtb39a; B, reference) (Dillon etc., Infect Immun 67:2941-2950,1999), Mtb8.4 (Coler etc., J Immunol161:2356-2364,1998), Mtb 9.9 (Alderson etc., J Exp Med 191:551-560,2000) (Coler etc., J Immunol 161:2356-2364,1998), Mtb 9.9 (Alderson etc., J Exp Med 191:551-560,2000), EsxG (Rosenkrands etc., Electrophoresis 21:3740-3756,2002), 19kDa antigen (Collins etc., J Gen Microbiol 136:1429-1436,1990), antigen 85b (Borremans etc., Infect Immun57:3123-3130,1989) (two kinds merge thing, A﹠amp; B, reference) peptide merges thing.Peptide is resuspended among the DMSO, and will reaches 50 kinds of peptides and be incorporated in one and merge in the thing, making the every kind of peptide concentration that merges in the thing is 1mg/ml.Peptide is merged thing to be stored under-80 ℃.

Clone and T cell clone.The B clone LCL that EBV transforms contributes plan (National Marrow Donor Program (NMDP from the supernatant generation of clone 9B5-8 (American type culture collection (American Type Culture Collection)) or from national marrow; Minneapolis, MN)) obtain.LCL is by keeping ((Heinzel etc., J Exp Med 196:1473-1481,2002) according to former description continuous passage., the DC that uses the Mtb infection separates the Mtb specific T-cells from the individuality of suffering from LTBI or active tuberculosis and clones as APC and the former limited dilution cloning method of describing (Lewinsohn etc., J Immunol 165:925-930,2000).In simple terms, instructions (Miltenyi Biotec according to manufacturer, Auburn CA) use the pearl of CD4 antibody sandwich to carry out negative screening, use the magnetic beads of CD8 antibody sandwich to carry out positive-selecting then, or by flow cytometry, from PBMC separation of C D8 ⁺The T cell.In this case, CD4-PE (BD Biosciences, catalog number (Cat.No.) 555347) is negative, CD8-APC (BD Biosciences, catalog number (Cat.No.) 555369) positive cells (purity＞99%) is stored on the Becton Dickenson LSR II.At 1X 10 ⁵Individual generation as described below irradiated under the existence of DC that body Mtb infects and rIL-2 (5ng/ml) is seeded in the T cell by 200 μ l with various concentration and augments in the cell culture medium that the RPMI 1640 of 10% human serum constitutes.The DC that uses ELISPOT and Mtb to infect originates as APC, to showed the hole assessment Mtb specificity of growth between 10-14 days.The specific T cell of Mtb is expressed at α β TXi Baoshouti by FACS and further phenotypic evaluation is carried out in the CD8 expression to having kept, and increases according to following description.The usage of V β is determined in use from the IOTest Beta Mark kit of Beckman Coulter.

The amplification of T cell clone.For the CD8 that increases ⁺The T cell clone, the rapid amplifying scheme (Heinzel etc., J Exp Med196:1473-1481,2002) of having used the anti-CD3mAb of utilizing of former description to stimulate.

The generation of peripheral blood DC and infection.Prepared monocyte derived DC (Heinzel etc., the same; Romani etc., J Exp Med 180:83-93,1994).In order to produce the DC that Mtb infects, (1X 10 with cell ⁶Individual) there is overnight incubation under the situation of Mtb (infection multiplicity [MOI]=50: 1).After 18 hours, harvesting also is resuspended in it in human serum of RPMI/10%.

The MHC binding analysis.Utilize MHC-peptide binding analysis to measure the peptide part and suppress the ability that radiolabeled peptide combines with the MHC molecule of purifying, and (Sidney etc. have been described elsewhere in detail, the Unit 18.3 in " 1999. immunology modernism " " measure the interaction (UNIT 18.3 Measurement of MHC/peptide interactions by gel filtration.In Current Protocols in Immunology) of MHC/ peptide by gel filtration; chief editors such as Coligan, John Wiley ﹠amp; Sons, Inc., 1996).In simple terms, with MHC molecule, test peptides and the radiolabeled probe peptide of purifying under the situation of the protease inhibitor cocktail that has human B2-microglobulin, incubation at room temperature.At incubation two days later, on the plate that the MHC/ peptide complexes is captured in W6/32 antibody (anti-HLA A, B and C antibody) or B 123.2 (antibody of anti-HLA B, C and some A) bag quilt, and use micro-scintillation counter measure per minute in conjunction with counting (cpm), measure radiolabeled peptide and combining of I class MHC molecule accordingly.For competitive analysis, calculate peptide concentration in conjunction with generation 50% inhibition to radiolabeled peptides.The peptide typical case is being covered under 6 variable concentrations of 100,000 multiple dose scopes, in three times or above independent analysis, testing.Under employed condition, as [label]＜[MHC] and IC ₅₀During 〉=[MHC], the IC that measures ₅₀Value is the rational approximate value of true Kd value.

IFN-γ ELISPOT analytical approach.IFN-γ ELISPOT analytic approach was carried out (Beckman etc., J Immunol 157:2795-2803,1996) according to former description.In order to measure CD4 to Mtb infects or Mtb antigen responds ⁺Or CD8 ⁺The stripped frequency of T cell, will use magnetic beads (Miltenyi Biotec, Auburn CA) from the PBMC positive-selecting to CD4 ⁺Or CD8 ⁺The T cell is property T cell source in response, carries out the double test under four kinds of different cell concentrations.Use as APC, merges thing (final concentration of every kind of peptide is 5 μ g/ml) pulse with DC with the Mtb infection or with peptide from body DC (20,000 cells/well), joins in the analysis then.For the analysis of using the T cell clone, with incubation under T cell (1000 or 5000 cells/well) and the situation that has or do not exist antigen from body LCL (20,000 cells/well).

Data analysis: in order to measure the stripped frequency of T cells with antigenic specificity, the spot average in each hole of each duplicate determination is mapped to the response cell quantity in each hole.Use linear regression analysis to determine the slope of straight line, the frequency of its expression T cells with antigenic specificity.If the binomial probability of amount of speckle (Lewinshon etc., Microbes Infect 8:2587-2598,2006) is obviously different with experiment and check analysis, then analyze and be considered to positive, promptly reflected the existence of the t cell response that is initiated.For the difference of the T cell frequency that exsomatizes between determining not on the same group, used the Wilcoxon/Kruskal-Wallis analysis.

Embodiment 2

Determine the Mtb specific C D8+ antigen of immunodominance

In order to determine the Mtb specific C D8 of immunodominance ⁺Antigen also determines that these are replied and whether comes from Mtb and infect, and used from not infecting, have LTBI or the CD8 of the donor that the Mtb activity infects being arranged ⁺The T cell.Reply direct isolated measuring, or use the CD8 that obtains by the limited dilution cloning on body DC (Lewinsohn etc., J Immunol 165:925-930,2000) that infects at Mtb ⁺The T cell clone is measured.Because the advantage CD4 that knows ⁺Mtb antigen is many, has therefore selected one group of these antigen of generally acknowledging further to assess.They are: Mtb39, CFP10 and Mtb8.4, Mtb9.9, ESAT-6, Ag85b, 19kDa and EsxG.For fear of the deviation that the peptide of the HLA binding specificity that uses prediction is introduced, synthesized the overlapping peptide (15aa, overlapping 11aa) (Lewinshon etc., J Immunol 166:439-446,2001) of representing target protein.

In order accurately to measure CD8 ⁺Stripped effector cell's efficient of T cell has been used linear regression analysis.As shown in fig. 1, in IFN-γ ELISPOT analyzes, at certain CD8 ⁺The interior CD8 of T cell quantity scope with the magnetic beads purifying ⁺The T cell is tested at the DC of peptide pulse.As described belowly carry out positive determining of analyzing, and, use linear regression to measure the antigen-specific resistant frequency if positive.

Assessed the object (n=12) that do not infect object (n=14), the object (n=20) of LTBI is arranged and suffer from activity TB to one group of Mtb CD4 ⁺The CD8 of T cellular antigens and DC that Mtb is infected ⁺Reply.All tested objects all have strong CD8 to the DC that Mtb infects ⁺T cell response, and in the individuality of suffering from activity TB with the higher (p=0.01 of individual specific strength mutually that LTBI is arranged; Fig. 2, Table I).But, find CD8 ⁺The T cell almost appears in the individuality of Mtb infection specially to replying of Mtb antigen group, because for 7 kinds in 10 kinds of antigen, aspect intensity of replying (Fig. 2) and the positive ratio of analyzing (Table I) two, noticed do not infect and individuality that Mtb infects between the significant difference of statistics.

Table I, to the CD8 of known TB antigen ⁺T cell response

^aDing Yi positive analysis in this article

But, CD8 between the individuality of suffering from activity TB and LTBI ⁺The difference of t cell response is not significant difference.Although observed strong CD8 at many tested antigens ⁺T cell response, but can notice several CD8 that strong Mtb instructs that have equally ⁺The object of t cell response does not have verifiable replying to many tested antigens.

These stripped frequency data have confirmed existence that the high frequency of many known Mtb antigens is replied, but do not illustrate the restriction allele in the target gene, minimum epi-position or dominance hierarchy.In order to address this problem, the DC that uses Mtb to infect has carried out human CD8 ⁺The limited dilution cloning of T cell (referring to Lewinsohn etc., J Immunol 166:439-446,2001), and produced two groups of classics and CD8 non-classical HLA restriction ⁺The T cell clone.Use the known CD4 of representative ⁺The peptide of antigen merges thing, can determine the clone's of HLA-Ia restriction antigentic specificity (Table II) in clone over half.

Table H, many CD8 ⁺The T cell clone is discerned known CD4 ⁺The T cellular antigens

At the single representative clone D466D6 detailed proof that stems from the object of suffering from activity TB this method.As shown in Fig. 3 A,, clearly antigentic specificity is defined as CFP10 at merging the clone being tested of thing pulse from body DC with one group of peptide.At every kind 15 mer peptides that constitutes CFP10 merging thing the clone is tested then, find that epi-position is included in CFP10 _1-15In (Fig. 3 B), synthesized every kind of possible 8,9,10 and 11 amino acid whose peptides and test reaction then, find antigen active (Fig. 3 C) between the 2-11 amino acids.Similarly, at lymphoblastoid cell lines (LCL) each clone has been carried out testing (Fig. 3 D) with total at least one the HLA type of donor.Total B4501 and C1601 from body LCL and IHW 9058LCL, present epi-position to the clone, the two is accredited as possible restriction allele with B4501 and C1601.But, C1601 ⁺D433LCL does not present epi-position, has eliminated C1601 as candidate's restriction allele.Therefore, D466D6 is subjected to the restriction of HLA-B4501.As among Fig. 4 by what confirmed in every kind of likelihood epi-position of wide concentration range build-in test, minimum epi-position is confirmed as CFP10 for D466D6 _2-10The experimental data of the appointment of supporting minimum epi-position is provided for each clone in the figure that replenishes.The summary of antigentic specificity, minimum epi-position and HLA restriction allele is presented in the Table III.Unexpectedly, except a kind of, every other T cell clone all is subjected to the allelic restriction of HLA-B.In addition, observing minority epi-position length is 9 amino acid.

The summary of Table III, the epi-position that identifies

Because each individual CD8 ⁺The T cell clone is based on that the growth of the DC that Mtb infects obtains, and therefore exsomatizes and has determined whether the antigen and the epi-position that identify have reflected the immunodominance epi-position.Taked two kinds of independently methods, whether first kind of definite response occurs with high frequency, and how many ratios determine for second kind has caused by epi-position in always the replying of antigen.In order to determine the effector cell's frequency that exsomatizes, as shown in Figure 1, use the CD8 that stems from the donor that separates the T cell clone from body DC and magnetic beads purifying ⁺The T cell is tested each epi-position.The summary of effector cell's frequency is presented in the Table III.For most applications, epi-position reflects that high frequency replys, and therefore can be taken as by being exposed to replying that Mtb causes.It should be noted that the T cell clone identification CFP10 that is separated to from four donors.For determine defined epi-position whether reflected considerable part at always the replying of target antigen, tested CD8 from the magnetic beads purifying of three donors with enough available peripheral blood lymphocytes (PBMC) ⁺The T cell merges thing to every kind of individuality 15 mer peptides, peptide and represents the reactivity of the peptide of minimum epi-position.As what confirmed in Fig. 5, the stripped frequency of minimum epi-position, 15 mer peptides that contain minimum epi-position and peptide merging thing is obviously consistent.These data show, obviously set up dominance hierarchy for each donor, and it are reflected among the original clone.At last, as what point out, often identify the identical filial generation clone of specificity in Table III, this result can arrive according to the immunodominance grade forecast.Use TCR V β dyeing to confirm that the clone between the filial generation clone concerns.What is interesting is that in two cases, consistent minimum epi-position and HLA restriction are by two different clone's representatives (Table III).

Because human CD8 at Mtb ⁺Many work of t cell response depend on uses the HLA prediction algorithm, therefore when having determined each epi-position, should inquire whether epi-position predicts by these methods.These epi-positions much do not have very strong hierarchical relationship.This may give prominence to these algorithms limitation in use.In order to address this problem by experiment, measured the IC of each synthetic in the process of the minimum epi-position of definition peptide at lineup's class HLA molecule ₅₀In Table III, shown IC for minimum epi-position with restriction allele of the same clan ₅₀Digital proof t cell epitope combine with HLA is strong, and between t cell epitope data and HLA binding data, demonstrate the height consistance.

Data acknowledgement, CD8 in the people that Mtb infects ⁺The frequency of t cell response and many common virus infect behind for example bovine vaccine, influenza and the cmv infection observed quite.The epi-position of all mappings except one, all is subjected to the restriction of HLA-B molecule.Data show, identify epi-position by using T cellular driven method, can determine the advantage epi-position in the people that Mtb infects.

Embodiment 3

At genome peptide library screening T cell clone

At the genome peptide library, screened the classical restricted and non-classical restricted T cell clone (referring to top Table II) that the known Mtb antigenic peptides of nonrecognition merges one of thing (Rv3875, Rv3874, Rv1886c, Rv0287, Rv3763, Rv1174c, Rv1196, Rv1793, Rv2346c, Rv1037c, Rv3619c and Rv1198).This peptide library has been represented 389 genes, represents Mtb genomic about 10%.For every kind of gene outcome, described peptide is 15 aggressiveness, has 11 amino acid overlapping.Each has synthesized every kind of peptide of 50nmol, is merged into the merging thing (9 blocks of plates) of 777 50 kinds of peptides then in 96 orifice plate forms.On every of 9 blocks of plates, comprised the hole of 5 blank well and an incoherent peptide merging thing SIV gag.For at genome peptide library screening and cloning, will clone at first amplification, and the DC that infects at Mtb tests, to guarantee that being cloned in ELISPOT from each of this specific amplification produces strong Mtb specific signals in analyzing.Then maximum 6 T cell clones are merged.In order to screen, with T cell clone (each clones 5,000 cells/well), merge thing (every kind of peptide 5ug/ml) from body DC (20,000 cells/well), IL-2 (0.5ng/ml) and peptide and in ELISPOT analyzes, under 37 ℃, be incubated overnight.Every kind of merging thing only carries out a technical repetition, because 5000 T cells in each hole and peptide antigen have produced inundatory positive findings, has obtained clear and definite result.Screened 6 classics clones at the genome peptide library, caused finding new epi-position from D504.This epi-position comes from the family of four kinds of albumen that comprise EsxJ, EsxW, EsxK and EsxP.These albumen have 98% homology, and difference has only 3 amino acid.There is the 5th member EsxM (Rv1792) in this family, and it is not included in the genome peptide library.

Each 15 aggressiveness that merges thing at these peptides comes screening and cloning.The clone of all 6 classics discerns EsxJ 21-35.This is EsxJ and other four zones that the member is consistent of this family.Next, prepared the peptide of 9,10 and 11 aggressiveness, and screened at each clone from this 15 aggressiveness.It is EsxJ 24-34 that minimum epi-position is confirmed as.In addition, find the restricted B5701 of being of HLA.

Embodiment 4

Additional screening T cell clone at the genome peptide library

Screened from 11 of D432B classical clones at above-described genome peptide library.Determine antigen for two clones, caused identifying two new epi-position PE_PGRS42 _47-55And PE9 _53-67The minimum epi-position of having determined a clone is PE_PGRS42 _47-55, and find the restricted B3514 of being of HLA.Another clone's minimum epi-position is also definite, but is included in the PE9 of 15 aggressiveness _53-67In.Find this clone's the restricted B3905 of being of HLA.

Table IV, from the details of the new epi-position of genome peptide library screening.

In the bracket clone's quantity of identification from the epi-position of each donor.* this is the protein family with almost consistent sequence.This family is made up of Rv1038c, Rv1197, Rv2347, Rv3620c.

The summary of Table V, the colony screening finished.

* the classics from D454 are cloned in amplification back nonrecognition Mtb again, and do not screen at the library.

* screens together from 426 and 431 classics clone, so only have a positive hole between these two clones.

Embodiment 5

At the stripped CD8 of genome peptide library screening ⁺The T cell

At the CD8+T cell of above-described genome peptide library screening from LTBI donor D610 (Southeast Asian).Every block of genome peptide library plate carries out the double screening, and each screening is 18 blocks of ELISPOT plates altogether.CD8 ⁺The T cell is from the CD8 of PBMC by using magnetic beads to separate of freezing preservation ⁺Screening prepares.The cell colony that obtains contains 〉=96% CD8 ⁺The T cell.Peptide in the ELISPOT plate (final concentration of every kind of peptide is 5ug/ml) adds CD8 ⁺T cell (250,000 cells/well), from body DC (20,000 cells/hole) and IL-2 (0.5ng/ml).On every block of plate, comprise 5 nutrient culture media control wells.For every block of plate, from each hole of this plate, deduct the mean value in these 5 holes, between plate, to carry out normalization.Then each technical the repeating on each plate is scored.If spot form unit (SFU) deduct cultivate datum hole mean value more than or equal to 10, and SFU is more than or equal to the twice of nutrient culture media mean value, then scored in the hole positive (Hudgens etc., J.Immunol.Methods 288:19-34,2004).This donor is replied the hole of four kinds of peptides comprising EsxJ, EsxW, EsxK and EsxP.Screen the CD8+T cell at every kind 15 aggressiveness that merges thing from these peptides then, and find that---same area of EsxJ, EsxW, EsxK and the EsxP that describes among the superincumbent embodiment 3---made and being replied only to EsxJ 21-35.

7 other donors are screened at the genome peptide library.Reply for the highest 10 and be described in detail in the table 7.Merging things with four kinds of outstanding peptides of yellow comprises from the peptide of a gene only.These four genes contain four new epi-positions.

Table V, merge the highest 10 of thing screening from the peptide of 7 donors and reply.It is at 250,000 CD8+T cells that spot forms unit.

Embodiment 6

Use the CD8+T cell detection in children, to diagnose TB

This result has proved use CD8 ⁺The T cell is diagnosed beat all sensitivity and the specificity of TB in children.

Method

Participator and program: will enlist in two clinical research groups from the different participators that enlist a little of Uganda Kampala.For resurrectionist (HE) group of health, the children (age was less than 15 years old) that contact with adult family use the AFB smear positive to assess, and the lung TB that cultivates checking is enlisted in the prospective cohort study of Uganda Kampala.In simple terms, seek to enlist after the TB nursing adult kinsfolk.When research is selected, on standardized tabular, collect detailed demography and clinical information, the standardization examination questionnaire of the symptom of activity TB is provided, and checks UP and shirtfront radiograph (CXR).When research is selected, write down all children's body weight and height.By the body mass index (BMI) of individuality and WHO children growth standard are compared to determine nutrition condition, wherein BMI Z-score value is-3 or the lower severe malnutrition that is defined as.Adopt the Mantoux method, (Pasteur M é r í eux Connaught, Swiftwater PA) carries out tuberculin skin test (TST) to the purfied protein derivative of 5 units of use.Test is carried out by nurse or well-trained medical worker, and is placing reading in 48-72 hour.Use WHO standard (WHO 2006) definition positive test, wherein scleroma is considered to positive greater than 5mm for the children of severe malnutrition, and scleroma is considered to positive greater than 10mm for all the other children.Can obtain all research participators' TST result.By ELISA all children more than 18 months have been carried out the HIV test; Only be found to be HIV for the age less than 18 months children and just carry out the HIV test when positive the biology parents.When enlisting or in 6 to 24 months observation period, have the related indication children of activity TB, accept research doctor's comprehensive clinical and diagnostic assessment, comprise the mycobacterium smear and the cultivation of CXR and at least one stomach aspirate sample of repetition.Sample is handled by conventional method, and the dyeing of experience fluorophore is with detection AFB, and cultivation is on the Loewenstein-Jensen nutrient culture media and in the Middlebrook 7H9 meat soup.AFB growth the carrying out monitoring in 8 weeks to all mycobacterium cultures.The microbiologist is unwitting to participator's the TB classification and the result of TST test.Only be included in the children that activity TB did not take place after 6 months.Having the past or the children of current TB history or the children of immunosupress (accept corticosteroid or suffer from HIV) is excluded.Obtained written Informed Consent Form.

For make a definite diagnosis+TB group that may (CP), enlisted and satisfied that WHO makes a definite diagnosis or possible TB standard (table 1; WHO 1983) acute ill children (≤10 years old).Hospitalized child (doubtful TB to sings and symptoms with TB, WHO 1983), just use comprehensive clinical evaluation, CXR, TST and HIV Enzyme Linked Immunoadsorbent Assay (ELISA) to assess if surpass two years old, if perhaps just used HIV polymerase chain reaction (PCR) to assess less than 18 months.The execution of TST and explanation and HE children's is identical.According to the result of this assessment, enlist the children that satisfy possible TB standard.Detailed demography and the clinical information of perspective collection on standardized tabular, and when bimestrial following up a case by regular visits to, by the research doctor LC is assessed.With identical to the HE children, record children's body weight and height, and assessment nutritional status.The children that enlist carry out the mycobacterium smear and the cultivation of the sputum sample that portion induces.Obtain the lymph node aspirate in some cases, be used for pathology and/or mycobacterium smear and cultivation.Follow up a case by regular visits to the TB that children accept to make a definite diagnosis, possible TB or do not have the final identification of TB according to bimestrial.The children that do not suffer from TB are excluded outside analyzing.Assigning the researchist of TB classification is unwitting for the result that ELISPOT tests.Before research is enlisted, obtained to adopt the written Informed Consent Form of local language from each children's parents or guardian.

When research is enlisted, before carrying out TST, extract 1-2cc/kg (20cc at most) blood from all children.By standard method separating periphery blood monocytic cell (PBMC), and with its cryopreservation.

Nutrient culture media and reagent: nutrient culture media is by having augmented 10% human serum, 5X 10 ^-5The RPMI 1640 of M 2ME (Sigma-Aldrich) and 2mM glutamine (GIBCO BRL) constitutes.Peptide is synthetic by Genemed Synthesis company.The single synthetic peptide that 15 aggressiveness that having synthesized has 11 amino acid (aa) overlapping representative Mtb specific proteins, CFP-10 and ESAT-6 constitute merges thing.Peptide is resuspended among the DMSO, and 43 kinds of peptides are merged into a merging thing, make that the concentration that merges every kind of peptide in the thing is 1mg/ml.Peptide is merged thing be stored in 8 ℃.

IFN-γ ELISPOT analyzes: the IFN-γ ELISPOT that spends the night according to former description (2) analyzes.Analysis is carried out on the PBMC of cryopreservation.Under the patronage of TBRU, associating Clinical Research Center (Joint Clinical Research Center (the JCRC)) Immunology Lab that the preparation of PBMC, cryopreservation and IFN-γ ELISPOT analyze at Uganda Kampala carries out.In order to determine ESAT-6/CFP-10 specific C D4 ⁺The frequency of T cell is used full PBMC property T cell source in response.In order to determine ESAT-6/CFP-10 specific C D8 ⁺The frequency of T cell, the CD8 that uses from PBMC the combination feminine gender with CD4 and CD56 magnetic beads (Miltenyi Biotec) to screen ⁺The T cell is property T cell source in response.Although found that with the dendritic cell (DC) that the monocyte of peptide pulse produces be the counting CD8 that exsomatizes ⁺The sensitiveest antigen presenting cell (3) of T cytological effect cell, but it needs enough PBMC to produce DC.For these research, available blood flow volume has been got rid of this method.Therefore, used the magnetic beads dilution to allow using endogenous monocyte as antigen presenting cell.In preliminary experiment, the CD4 dilution has produced high background, and it can be by the CD56 of dilution simultaneously ⁺The NK cell is eliminated.When carrying out directly comparing with use DC, this method is at counting antigentic specificity CD8 ⁺The efficient aspect of T cell is about 80%.Flow cytometry shows, CD4 pollution rate＜2%, and CD8 purity＞85%.All the other cells are mainly by monocyte and B cellularity.Use the PBMC (CD4 of 250,000 cells/well ⁺The T cell analysis) or the PBMC (CD8 of CD4/CD56 dilution ⁺The T cell analysis), and uses peptide to merge thing, carry out IFN-γ ELISPOT as antigen source (final concentration of every kind of peptide is 5 μ g/ml).Comprise feminine gender and positive control in each is analyzed, it still comprises phytohaemagglutinin (PHA, 10lg/ml by containing cell and not containing antigen or do not contain antigen respectively; EMDBioscience) hole constitutes.All mensuration are carried out two parts of parallel samples.In some cases, carry out the parallel control of three parts of no antigens (nutrient culture media).

In order to measure the stripped frequency of T cells with antigenic specificity, measure the average that spot forms unit (SFU) for each hole of each two parts of parallel sample, and compare with SFU average in the nutrient culture media contrast.For the changeability between parallel sample mesopore of interpretation technique and the hole, calculated the standard deviation of nutrient culture media contrast.Positive ELISPOT analyzes and is defined as wherein antigentic specificity and replys the analysis that is higher than at least two standard deviations of background contrast.If satisfy this standard, subtracting background is replied to determine antigentic specificity.Positive PHA replys and is defined as each hole 〉=30SFU.

Research and design and statistical study: carried out cross-sectional study and be used for blood is extracted in comparison out from baseline CD4 ⁺And CD8 ⁺T cell response, and to two clinical research groups---the children or the HE children that suffer from CP-TB compare.In analyzing for the first time, be independent of the CP-TB group HE seminar is studied, to study the age to the influence that the Mtb specific T-cells is replied takes place.For this analysis, all children of≤15 years old have been studied.Next, for CP-TB and HE seminar are compared, the children that only select≤10 years old from the HE group are to adjust age differences inherent in the group, and this is because CP-TB seminar only recruits age≤10.

ELISPOT is analyzed data (SFU), and (WA USA) is input in the SAS data file for Microsoft CORP, Redmond, and all analyze use SAS 9.1 editions, and (NC USA) carries out for SASInstitute Inc, Cary from Excel.Baseline single argument between HE and CP-TB and the TB (C-TB) that makes a definite diagnosis relatively uses students t check to carry out for continuous variable, uses Chi-square Test (maybe use fishers rigorous examination when specifying) to carry out for classified variable.Similarly, the classification of the frequency that the positive ELISPOT that is undertaken by the clinical research group analyzes relatively uses Chi-square Test to assess.The nonparametric analysis (wilcoxon rank test) that use is used for continuous variable relatively is higher than the SFU of background.Sensitivity is calculated as the quantity from the total middle-jiao yang, function of the spleen and stomach analysis of explainable analysis of independent CP-TB group or C-TB group.Specificity is calculated as the quantity that feminine gender is analyzed in the explainable analysis sum in the HE group.

In order to study the factor relevant with CP-TB, several models are assessed, with research to possible when mixing covariant and adjusting, positive ELISPOT analyze to the influence of the relevance of clinical research group.Thus, according to CD8ELISPOT analyze, CD4ELISPOT analyzes, age (0-5,5-10 year), nutritional status (BMI) and TST result's explanation, the probability that will be in the CP-TB clinical research group in organizing with respect to HE is set up model.At first, independent CD8 and CD4ELISPOT predicted value are checked in following model: (1) log probability (clinical research group)=alpha+beta ₁(+CD8ELISPOT/-CD8ELISPOT)+β ₂(age)+β ₃(ZBMI)+β ₄(TST); (2) log probability (clinical research group)=alpha+beta ₁(+CD4ELISPOT/-CD4ELISPOT)+β ₂(age)+β ₃(ZBMI)+β ₄(TST).In two kinds of models, the clinical research group of reference is the HE group.The predicted value of CD8 and CD4ELISPOT analysis is assessed in same model.For this, following model is carried out match: log probability (clinical research group)=alpha+beta ₁(CD8ELISPOT)+β ₂(CD4ELISPOT)+β ₃(age)+β ₄(ZBMI)+β ₅(TST), wherein reference clinical research group remains the HE group.On all models, carry out backward logistic regression then to increase model fitting.

The result

In order to study the age to CD4 among the HE children ⁺And CD8 ⁺The influence of t cell response, (Fig. 9 a) to have assessed the contact children of family of 129 age≤15 year old.Get rid of and to have comprised that 20 are enlisted children and 5 children that are found to be the HIV positive that TB takes place in back 6 months at baseline.Therefore, the contact children of family of 104 age≤15 year old have been carried out the ELISPOT analysis, and comprised in final analysis that 98 PBMC ELISPOT analyze and 79 CD8ELISPOT analyses.In order to compare the CD4 between HE group and the CP-TB group (≤10 years old) ⁺And CD8 ⁺T cell response, the ELISPOT that has only comprised from the HE children of age≤10 year old analyzes data.For this comparative analysis, (Fig. 9 a) to have comprised the ELISPOT analysis of carrying out on 62 HE children.For CP TB group, assessed the negative children's of HIV (Table VI) of 101 doubtful TB qualification.Wherein, having enlisted 96 has the children of that make a definite diagnosis or possible TB and has carried out CD4 and CD8ELISPOTS, and it is explainable having 82 PBMCELISPOT and 87 CD8ELISPOTS to analyze respectively, and is included in (Fig. 9 b) in the final analysis.Expose children at the TB of health, the children of that make a definite diagnosis or possible TB are arranged and have between the children of the TB that makes a definite diagnosis, do not have significant difference between the quantity of explainable sample.。

Table VI

The clinical research group (HE (≤10 years old) and CP-TB) in all children of enlisting Clinical symptoms and relatively be presented in the Table VII.

The baseline characteristic of Table VII, HE and CP-TB seminar.Use students t check (ζ) Satterthwaite unequal variance to calculate for the p value of continuous variable report.Use card side's method to calculate for the p value of grouped data (TST and sex)

The children that suffer from CP-TB compare malnutritive (p＜0.001) and more young slightly (0.01) more with HE group.In HE and CP-TB children, the frequency of positive TST is suitable.The children of the children (C-TB) that the TB that makes a definite diagnosis arranged and HE children (p＜0.001) and possible TB (P-TB, p=0.01) compare malnutritive more, but aspect age, sex or the TST result and HE children or have the children of P-TB not have difference.Only the children's that explainable ELISPOT result is arranged that enlist in HE (≤10 years old) and CP-TB group clinical research group baseline clinical characteristics (age, sex, BMI and TST state) does not have difference with all features of enlisting children.

At first, reply, in HE group, analyzed the intensity that the Mtb specific T-cells is replied, and＜5 years old children and 5≤15 years old children are compared for the Mtb specific T-cells that relatively in children, obtains in time.In two age groups, all observed powerful CD4 ⁺T cell response, but in the children of age＜5 year old, compare CD8 with the juvenile ⁺The t cell response reduction (p=0.055, Figure 10).These data acknowledgements in the child, lack CD8 ⁺T cell response.

Next, the HE children and suffer from and compared the Mtb specific T-cells between the children of TB and reply.Compare with HE group, (a) and in all suffer from the children of TB (CP-TB group, p=0.008), the ratio that positive CD8ELISPOT analyzes is higher for C-TB group, p=0.001, Fig. 3 in the children that suffer from the TB that makes a definite diagnosis.The ratio that positive CD4 (PBMC) ELISPOT analyzes is higher in than HE group in C-TB group (p=0.02, Fig. 3 a), but between CP-TB and HE group suitable (p=0.14).By age stratification, HE, C-TB and CP-TB group are compared then.Similar to analysis result from all children, when only HE children and the age of suffering from TB being compared less than 5 years old children, the ratio that positive CD8ELISPOT analyzes is in the children that suffer from the TB that makes a definite diagnosis (C-TB group, p=0.009, Figure 11 b) in and suffer among the children (CP-TB group) of TB higher at all.But when the children that only consider＜5 years old, the ratio that positive CD4 (PBMC) ELISPOT analyzes between all groups is (Figure 11 b) quite.In 5≤10 years old children, the ratio that positive CD8 and CD4 (PBMC) ELISPOT analyzes is high in than HE group in CP-TB group, but suitable between C-TB and HE group.

Although the assessment of the test performance of CD4 and CD8ELISPOT Analysis and Identification TB is subjected to the little restriction of group size, but still carried out sensitivity and the specific exploratory analysis that positive ELISPOT analyzes, wherein use C-TB to be respectively applied for meter sensitivity and specificity as golden standard TB group and HE group.In the children of age≤5 year old, the sensitivity that CD4 and CD8ELISPOT analyze is that suitable (be respectively that C-TB is 56% (CI 0.30-0.78), C-TB is 47% (CI 0.24-0.71).But CD8ELISPOT analyzes than the CD4ELISPOT analysis and has more specificity (being respectively 88%CI 0.68-0.97 and 62%CI 0.44-0.78).

In 5＞10 years old children, the sensitivity similar with specificity (sensitivity, CD4100%[(CI 0.47-1.0) of CD4 and CD8ELISPOT analysis], CD8,86%[(CI 0.0.42-0.99)]; Specificity, CD463%[CI 0.40-0.82], CD870%[CI 0.45-0.88]).Can put question to which variable to influence then and/or obscure the positive or negative ELISPOT that strides age level.Carried out the logistic regression analysis so that set up model, and comprised CD8 and CD4ELISPOT, age, nutrition condition (Z value/BMI) and baseline TST state for the covariant relevant with CP-TB.For preceding two models, the covariant of positive independently CD8 and CD4ELISPOT is set up model, then together iteration in the 3rd model of Table VIII.

Table VIII: the multivariate logistic regression of ELISPOT analysis result is analyzed *

* according to the various covariants that show among the model 1-3 log probability that make a definite diagnosis or possible TB is arranged is set up model.In model 1, in being arranged, the children of positive CD8ELISPOT have the probability of that make a definite diagnosis or possible TB high 3.8 times (the Hosmer Lemeshow goodness of fit is 0.07).On the contrary, as shown in model 2, CD4ELISPOT uncorrelated with TB that make a definite diagnosis or possible (Hosmer Lemeshow goodness of fit p=0.15).Be used for the two the model 3 of covariant of CD8 and CD4ELISPOT comprising, in the children that have at the adjusted positive CD8ELISPOT of other covariants in the model, there is the probability of that make a definite diagnosis or possible TB high 4.7 times (the Hosmer Lemeshow goodness of fit 0.21).

Children with positive cd8 t cell ELISPOT with age, BMI and the adjusted health of baseline TST are compared with the children that exposed, have the probability (p=0.004) of high 3.8 times trouble CP-TB.In contrast to this, the children with positive CD4ELISPOT do not have the probability of higher trouble CP-TB.In the model that comprises CD8 and CD4ELISPOT covariant, after CD4ELISPOT result is adjusted, the existence of positive CD8ELISPOT with suffer from the CP-TB significant correlation.In order to increase the match of model, used logistic regression backward.In this model, after adjusting at the age, the probability of in having the people of positive CD8ELISPOT, suffering from that make a definite diagnosis or possible TB be healthy exposure group the people 4.6 times (CI 1.8-12.1) (p=0.002).Cd4 t cell ELISPOT does not increase the overall match of model, and is being eliminated (Hosmer Lemeshow goodness of fit p=0.68) with BMI and TST state in the iteration selection course backward.

CD8 between the clinical research group ⁺And CD4 ⁺The intensity of t cell response (Figure 12).For the children of age≤5 year old, CD8 in suffering from the children of TB ⁺The intensity of t cell response big (CP-TB, p=0.01; C-TB, p=0.009), and CD4 ⁺T cell (PBMC) is replied between clinical group suitable.Similarly, for the children of age at 5≤10 years old, CD8 between HE, CP-TB and C-TB group ⁺And CD4 ⁺The intensity of t cell response is suitable.

Embodiment 6

The diagnosis of the outer TB of lung

The diagnosis of the outer TB of lung is challenging especially.Outside suffering from lung, studied CD8+T cell response among Uganda children of TB at ESAT-6 and CFP-10.Among the children of TB, it is positive that 51% CD8ELISPOT analyzes outside suffering from lung.In addition, CD8 among the children of TB outside suffering from lung ⁺The intensity of t cell response and the children that suffer from intrathoracic TB suitable (Fig. 8).Therefore, the test based on cd8 t cell can be used for diagnosing the outer TB of lung.

Embodiment 7

The clinical testing of extensive checking property

A. study the participator

The hospitalized child of age＜5 year old is enlisted in clinical testing.The children that juvenile and HIV infect get rid of from research.Research and design be used for the comparison children (n=80 may+TB that makes a definite diagnosis; The TB that make a definite diagnosis n=～20) with (n=50) group of the children (LRTInotTB) that suffer from the lower respiratory infection of non-TB.The initial identification of intrathoracic TB uses WHO to be used for carrying out in the criterion of children's tentative diagnosis TB, and enlists the children of the intrathoracic TB that suffers from possibility.The summary of these criterions is presented in the table 1.Specifically, use the result of clinical medical history, TST and CXR to carry out the tentative diagnosis of possible TB.Other inclusion criterias comprise the TB treatment that is shorter than month.When enlisting latter two month, use Clinical Follow-up, comprise the response of antagonism TB treatment and get rid of other diagnosis and the Mtb cultivation results, make the TB that makes a definite diagnosis, possible TB or the final identification of non-TB.Regarding as the children that suffer from that make a definite diagnosis or possible TB when following up a case by regular visits in 2 months will stay in total intrathoracic TB group.The children that do not suffer from TB in the time of two months will get rid of from analyze.Because can not require for research is selected that therefore Mtb cultivates checking by cultivating TB case＜40% that confirms diagnosis.But for data analysis, the subgroup of suffering from the TB that makes a definite diagnosis of intrathoracic TB group (cultivating the intrathoracic TB of checking) has been represented the main comparison with the non-TB of LRTI group.The non-TB of LRTI group is defined as by unusual CXR and compatible symptoms of pneumonia and the children of the determined LRTI of suffering from of sign.In addition, this group must not have as defined doubtful TB in the table.

The non-TB of LRTI group experience and the identical clinical and laboratory study of intrathoracic TB group.Similar to group, the Clinical Follow-up in the time of 2 months is used to make the final identification of the non-TB of LRTI.Cultivate the positive if Mtb in any of these children, unexpectedly occurs, so these children are got rid of from the analysis of the non-TB group of LRTI.

For the research that is proposed, identify age＜5 year old and have the children of LRTI sings and symptoms.Understand medical history and check UP, and obtain CXR, and the HIV The selection result is checked.The children with positive HIV ELISA result of age＜18 month need HIV PCR to test to confirm to infect.All have the serological children of negative HIV, and age＜18 month have positive HIV ELISA but the children of negative HIV PCR can comprise under study for action.Have the serological children of positive HIV, comprise＜18 months and the children that do not have available HIV PCR test result or have positive HIV PCR test result, from research, get rid of.Enlist that HIV is not infected, age＜5 year old, as to satisfy the standard of intrathoracic as defined above TB or the non-TB of LRTI children.All experimenters experience that TST disposes and the sputum that is used for AFB smear and cultivation is induced.TST uses protein derivatives (PPD, 5TU, the Tubersol of purifying; Connaught Laboratories, Limited, Toronto, Canada) and the Mantoux method carry out.The sputum that is used for AFB smear and cultivation is induced.The experimenter draws blood when enlisting, and separation＞25X 10 ⁶Individual PBMC is used to finish the research of all 5 kinds of antigen combinations.In the time of two months, recall the experimenter and carry out the follow-up investigation prescription on individual diagnosis.In this time gone to a doctor, to the experimenter mid-term medical history and laboratory result check.Carry out final identification (the non-TB of intrathoracic TB and LRTI) this moment to research group.

The data management of B. demographic, clinical and immunology data

CWRU TBRU data management infrastructure or other similar data supervisory routines are used in this research.For example, can use the TELEform that provides site automaticdata from afar to login ^TMV5 Elite software (Cardiff Software, San Marcos California).In simple terms, formulate data collection list, and make its form and TELEform by ugandan data manager ^TMThe software butt joint.After running into the patient, the portion of data form copy is placed in the clinical chart, another part form is sent to local data center, is scanned into TIF (tagged image file format) image file of multipage there.With these File Compress and storage.Then the TIF data file is read in TELEform ^TMProgram, this program is arranged form and record data according to predetermined electronic stencil, then data is sent in the data base management system (DBMS).In case after entering electronic databank, use standard program to edit and clear up data, with the data that mark disappearance and the value of super scope.Produce formal inquiry from data center, local data manager solves inquiry, revises database and record variation.Electronic data backs up, for example backup every day.

Demography relevant with this research and Clinical symptoms comprise the feature about age, sex, disease description, HIV serology state, BCG inoculation state, the body weight at this age, the height at this age, TST result and Mtb cultivation results.In addition, carry out evaluation of nutrition, and the z value with respect to the body weight of age and height is calculated for all children that enlist.At last, for each experimenter assigns unique identiflication number, be used for database.

C. in the children that suffer from intrathoracic TB and the non-TB of LTBI, there is Mtb antigens c D8 ⁺The T cell

CD8 ⁺T cell response uses IFN-γ ELISPOT to analyze and measures, and the PBMC that uses CD4 and CD56 dilution is as antigen presenting cell (APC) and response CD8 ⁺The source of T cell.Specifically, carrying out CD8ELISPOT on the PBMC of cryopreservation analyzes.Although the DC with the peptide pulse exsomatizes to cause CD8 ⁺Sensitive and the most special means of t cell response, but it needs enough PBMC with generation DC, and highly purified CD8 ⁺The T cell.For these research, the ability that available blood flow volume and execution longer-term are cultivated (DC) is limited.A kind of optional method is dilution CD4 ⁺The T cell, and use from the body monocyte as APC.In preliminary experiment, the CD4 dilution of PBMC has produced high background, and it can be by dilution CD56 ⁺The NK cell is eliminated.When directly comparing with use DC, this method is at counting antigentic specificity CD8 ⁺The efficient aspect of T cell is about 80%.Therefore, in order to measure CD8 ⁺T cell response has used the PBMC (250,000 cells/well) with magnetic beads dilution CD4CD56 cell in IFN-γ ELISPOT analyzes.Represent the synthetic peptide of two antigens combination to merge thing (15 aggressiveness, have 11 amino acid overlapping) and be used as the antigen source.Two antigen combinations are represented by 43,50,72,72 and 72 kind of peptide of being used for CFP10/ESAT6, CFP10/EsxJ, CFP10/PPE51, CFP10/CFPF, CFP10/PPE15 combination.As a result, the PMBC that uses as few as 10,000,000 cryopreservation has determined CD8 ⁺The T cell is replied 5 kinds of CD8 antigen combinations, and this needs the 1-5ml whole blood.It should be noted that for the blood flow volume that needs than young child lessly, this is because the every 1ml whole blood of baby's blood produces nearly 10,000,000 PBMC, and produces 1,000,000 PBMC of 1-2 from the every 1ml of juvenile and adult's blood.If experimental port deducts SFU in contrast (nutrient culture media) hole greater than 2 times of the standard deviation of control wells, analyze and be considered to positive.Then the intensity of replying is expressed as SFU/250,000 cell.As the contrast of the usefulness of magnetic beads dilution, the cell surface dyeing by CD4 is also used flow cytometry, has measured contaminative CD4 ⁺The percent of T cell.Any ELISPOT analyzes, if CD4 ⁺The percent of T cell surpasses 5%, and it is invalid just to think.

D. in the children that suffer from intrathoracic TB and the non-TB of LTBI, there is Mtb antigens c D4 ⁺The T cell? how is the intensity of comparing positive frequency of analyzing and positive response between the group?

For with CD8 ⁺T cell response compares, the CD8 that used dilution ⁺The PBMC of T cell is property CD4 in response ⁺The source of T cell, and use remaining cell to carry out CD4 as APC ⁺ELISPOT is as Mtb antigentic specificity CD4 ⁺The tolerance of t cell response.This and use PBMC, ESAT6/CFP10 peptide .TB and IFN-γ ELISPOT analyze closely similar analysis.Use the magnetic beads dilution cryopreservation PBMC of cd8 cell (250,000 cells/well) as CD4 ⁺IFN-γ ELISPOT has been carried out in the two source of T cell and monocyte/APC.The synthetic peptide identical with being used for the CD8 analysis merges thing and is used as the antigen source.CD4 to 5 kinds of antigen combinations ⁺T cell response, the PBMC that can use as few as 300 ten thousand cryopreservation determines.If experimental port deducts SFU in contrast (nutrient culture media) hole greater than 2 times of the standard deviation of control wells, analyze and be considered to positive.Then the intensity of replying is expressed as SFU/250,000 cell.As the contrast of the usefulness of magnetic beads dilution, the cell surface dyeing by CD8 is also used flow cytometry, has measured contaminative CD8 ⁺The percent of T cell.Any ELISPOT analyzes, if CD8 ⁺The percent of T cell surpasses 5%, and it is invalid just to think.

E. the TST positive as a result in the children that suffer from intrathoracic TB and the non-TB of LTBI

For with CD8 ⁺T cell response compares, and uses aforesaid standard method to carry out TST.Use WHO standard, positive TST is defined as scleroma 〉=5mm for severe malnutrition children (Z value＞-3), scleroma 〉=10mm for all the other children.

F. statistics is considered: the sensitivity of two antigen combinations and specificity and selection are used for two kinds of combinations of three kinds of Mtb antigen combination research (SA 2).

Main terminal point is CD8 ⁺T cell response, CD4 ⁺T cell response and TST result.CD8 ⁺T cell response and CD4 ⁺T cell response uses ELISPOT to analyze by the T cell that produces IFN-γ and measures.Main terminal point is the graded response that is defined as the adjusted ELISPOT counting of background.The adjusted ELISPOT counting of background defines according to the standard of being set up in the past by our laboratory (CD8 antigen is found plan).TST result analyzes as just the binary terminal point.

For main terminal point, used recipient's operating characteristic (ROC) curve method, and estimation ROC area under a curve (AUC) is as the tolerance of diagnostic accuracy.For every kind of incompatible theory of antigen group, whether tested AUC apparently higher than 50%, promptly whether there is the sign of any diagnostic utility.Determined the suitableeest cut off for the adjusted ELISPOT counting of background, so that provide higher sensitivity to keep suitable specificity simultaneously.Relatively carried out the ROC analysis for following one-level: the intrathoracic TB and the non-TB of LRTI that cultivate to confirm, and total intrathoracic TB (may+make a definite diagnosis TB) and the non-TB of LRTI.In addition, there is the non-TB of LRTI of LTBI to be defined as having in the non-TB of the LRTI group experimenter of positive TST.Then following secondary has relatively been carried out the ROC analysis: intrathoracic TB that cultivate to confirm and the non-TB of LRTI that LTBI is arranged, and total intrathoracic TB (may+make a definite diagnosis TB) and the non-TB of LRTI that LTBI is arranged.Use morbid state as a result of, the antigen response result has been set up Logic Regression Models as covariant (and any other potential obscure factor).Antigen reply can be used as binary and continuously covariant assess.Indication combination and other combinations of adding once only comprise one, whether significantly improve prediction to assess them.In addition, can carry out the suitableeest independent antigen group that program progressively selects to predict the disease result sets up jointly.The result of these analyses is weighted at main standard.

The sample size of 80 total intrathoracic TB (may+make a definite diagnosis TB) and 50 non-TB of LRTI allows the AUC that detects to increase by 15% (50% to 65%), and weighing (power) is 84%, and level of significance is 5%.Cultivate the intrathoracic TB of confirmation and the sample size of 50 non-TB of LRTI for 20, allowing to detect AUC increases by 20% (50% to 70%), and power is 77%, and level of significance is 5%.The increase of 15-20% is consistent with the preliminary data that proposes above.

The result: the antigen combination of identifying in this article (ESAT6/CFP10) in intrathoracic TB group to CD8 ⁺T cell, CD4 ⁺The T cell has similar result with TST, is about 50% positive the analysis.In the non-TB of LRTI group, do not detect CD8 at these antigens ⁺T cell response.Because other four kinds of antigens combinations also contain second immunodominance CD8 antigen except CFP10, therefore in intrathoracic TB group, observed at other CFP10/Mtb antigen combinations and compared CD8 at CFP10/ESAT-6 ⁺The frequency of T cell analysis increases.In the non-TB group of LRTI, do not detect CD8 at any tested antigen combination ⁺T cell response.CD4 at all antigen combinations ⁺T cell analysis and TST this group about 30% in be positive.In the intrathoracic TB group that cultivate to confirm, compare positive CD8 with whole intrathoracic TB group ⁺And CD4 ⁺The ratio of t cell response is close or higher.

Embodiment 8

Animal model

In tuberculosis research, mouse model has been widely used in simulating the various aspects of disease.Mouse can infect via various approach, comprises in intravenous, the peritonaeum and tracheae.A kind of approach is the aerosolized of biosome, is used for respiratory infections.Mouse is exposed to gasoloid (infect whole health or only infect nose) in cell.Dosage of the present invention can change by concentration or the exposure duration of handling Mtb in the sprayer.Infect by the gasoloid low dosage, for example about 50 colony-forming units (CFU) cause slowly stable increase of bacterial number in the lung, generally reach peak value in all around, and this conforms to the number of peaks of T cell in the lung.The acute phase that initial period is considered to infect.After infection, there be the diffusion of bacterium to mediastinal lymph nodes.Generally can detect the T cell between two to three weeks causes.After around approximately, it is stable that bacterial number reaches, and the pathology response of slowly development occurs.This system can be used for the simulation game sexuality and dyes.

The ability that can use method assessment objective composition described herein in animal model, to prevent infections.The efficient of objective composition can be made the CD8+ that replys or the quantity of CD4+T cell is monitored to the Mtb polypeptide by measuring in t cell response, the biological example sample.Analyze for these, T cell (T cells with one) is contacted with at least a mycobacterium polypeptide and the antigen presenting cell of presenting one or more mycobacterium polypeptide.The mycobacterium polypeptide comprises following shown amino acid sequence: (a) one of shown amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, EQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 or SEQID NO:12; Or (b) at least 9 to 20 continuous amino acid of the shown at least a amino acid sequence of SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ IDNO:10, SEQ ID NO:11 or SEQ ID NO:12, wherein said 9 to 20 continuous amino acid specificitys are in conjunction with the main histocompatibility complex of I class (MHC).Determine whether specific recognition mycobacterium polypeptide of T cell.The increase of the T cell quantity of specific recognition Mtb polypeptide shows that composition is effective.

Below to exemplary animal model be described (also referring to Repique etc., Infec.Immun.70:3318-3323,2002, this as additional aspects draw be with reference to):

A. short-term mouse model:

Use composition that the C57BL/6 mouse is carried out immunization campaign according to the scheme that is fit to, it was had a rest for 4 to 6 weeks.Use the low dosage gasoloid (50-100CFU) of the Much's bacillus that virulence is arranged to infect the mouse of immunity, and assess protective effect by the bacillus quantity that assessment in 30 days behind excitation is lived.

By with organ homogenate and with 10 times of continuous dilution rate bed boards on the 7H11 agar plate, lungs and the spleen of mouse carried out count plate.Dull and stereotyped incubation is reached 21 days most, and determine the colony forming single-digit of every kind of organ.

When comparing with the mouse that PBS handles, the premunitive mouse of BCG has the protective effect of about 1Log10 in its lungs and spleen.

Biological sample obtains before the administration objective composition He after the administration objective composition.Alternatively, biological sample obtains from the animal of media processes and the animal of objective composition processing.The increase of the T cell quantity that combines with Mtb polypeptide disclosed herein shows that composition is effective.

B. short-term guinea pig model

Use comprises the composition of the polynucleotide of one or more Mtb polypeptide or these one or more polypeptide of encoding, and the Hartley cavy of outbreed is carried out immunization campaign, and it was had a rest for 8 to 10 weeks.Use the low dosage gasoloid (10-30CFU) of the Much's bacillus that virulence is arranged to infect the cavy of immunity, and assess protective effect by the bacillus quantity that assessment in 30 days behind excitation is lived.

By with organ homogenate and with 10 times of continuous dilution rate bed boards on the 7H11 agar plate, lungs and the spleen of cavy carried out count plate.Dull and stereotyped incubation is reached 21 days most, and determine the colony forming single-digit of every kind of organ.Also obtain lungs and spleen and partly be used for histologic analysis.

When comparing with the cavy that PBS handles, the premunitive cavy of BCG has about 2-3Log in its lungs and spleen ₁₀Protective effect.In addition, when comparing with nonvaccinated animal, the premunitive cavy of BCG has the sharp outline granuloma.

Biological sample obtains before the administration objective composition He after the administration objective composition.Alternatively, biological sample obtains from the animal of media processes and the animal of objective composition processing.The increase of the T cell quantity that combines with Mtb polypeptide disclosed herein shows that composition is effective.

C. long-term guinea pig model

Guinea pig model and mouse model are similar, but experiment is open result's a survival type, can last up to 2 years.Cavy develops and human similar " classics " granuloma of suffering from active tuberculosis (TB), and when the downright bad development of lung tissue, they lose weight and die from TB to human similar the beginning.Can assess the colony forming single-digit in lungs and the spleen.Also can carry out histological examination and involve the degree with disorganization to determine lung.After the low dosage gasoloid exposed in cavy, the quantity of biosome increased gradually during first three week, reaches platform then and enters chronic states.The increase of bacterial loads in the lung appears in the late stage infecting, and this worsens relevant with pathological condition.Do not treat, the common rising of CD4 and cd8 t cell in the lung that infects cavy, will occur.

Use experimental vaccine that the Hartley cavy of outbreed is carried out immunization campaign according to the scheme that is fit to, it was had a rest for 8 to 10 weeks.Use the low dosage gasoloid (10-30CFU) of the Much's bacillus that virulence is arranged to infect the cavy of immunity then.To cavy weigh in weekly and every day monitoring of diseases sign (for example accelerated breathing and can not well grow).Nonvaccinated cavy 20 to 25 weeks behind excitation are died from infection, and the cavy of BCG inoculation survived behind excitation for 50 to 55 weeks.

When ptomatopsia, the CFU quantity and the pathology degree of lungs and spleen are assessed.Relative protective effect with the animal comparative experiments composition of BCG inoculation.

Biological sample obtains before the administration objective composition He after the administration objective composition.Alternatively, biological sample obtains from the animal of media processes and the animal of objective composition processing.The increase of the T cell quantity that combines with Mtb polypeptide disclosed herein shows that composition is effective.

Obviously, the accurate details of described method and composition can be changed or revise, and does not deviate from the spirit of described invention.We are to all such modifications of the scope and spirit that belong to claims and change prescription.

Claims (29)

1. method that is used for detecting human subjects Much's bacillus (Mycobacterium tuberculosis), it comprises:

From suffering from human children lungy or from doubtful biological sample separation of C D8+T cell with human subjects of Much's bacillus latent infection from doubtful; And

The CD8+T cell is contacted with one or more mycobacterium polypeptide;

Determine whether specific recognition mycobacterium polypeptide of CD8+T cell, wherein the existence of the T cell of specific recognition mycobacterium polypeptide detects the Much's bacillus in the object, thereby children are accredited as suffer from tuberculosis, or object is accredited as has the Much's bacillus latent infection.

2. the process of claim 1 wherein age of children less than 5 years old, or wherein children's age is 5 to 10 years old.

3. the process of claim 1 wherein that children are babies.

4. the process of claim 1 wherein that object is doubtful has a Much's bacillus latent infection.

5. the process of claim 1 wherein that children are doubtful suffers from pulmonary tuberculosis.

6. each method of claim 1 to 4, wherein the doubtful lung with Much's bacillus of object or children infects outward.

7. the method for claim 6, wherein outer infection of lung comprises lymphnoditis, tuberculosis of pleura, bone and joint tuberculosis, tuberculosis of central nervous system, abdominal tuberculosis, miliarytuberculosis or tuberculous pericarditis.

8. each method of claim 6-7, wherein to as if prepuberal teenager.

9. each method of claim 1-8 determines wherein whether specific recognition mycobacterium polypeptide comprises the measurement cytokine expression to the CD8+T cell.

10. the method for claim 9, wherein cell factor is interferon-(IFN-γ).

11. the method for claim 10 is wherein measured the expression of IFN-γ and is used specificity to measure in conjunction with the antibody of IFN-γ.

12. each method of claim 1-11, wherein said one or more mycobacterium polypeptide comprise following shown amino acid sequence:

(a) one of shown amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ IDNO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:39 or SEQ ID NO:61; Or

(b) at least 9 to 20 continuous amino acid of shown at least one amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:7, SEQ IDNO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:39 or SEQ ID NO:61, wherein said 9 to 20 continuous amino acid specificitys are in conjunction with the main histocompatibility complex of I class (MHC); And

(c) one of shown amino acid sequence of SEQ ID NO:39-83.

13. each method of claim 1-11, wherein the mycobacterium polypeptide comprises the shown amino acid sequence of SEQ IDNO:39.

14. each method of claim 1-11, wherein the mycobacterium polypeptide comprises the shown amino acid sequence of SEQ IDNO:61.

15. each method of claim 1-11, wherein the mycobacterium polypeptide comprises 9 to 20 continuous amino acids of the shown amino acid sequence specificity of SEQ IDNO:39 in conjunction with the main histocompatibility complex of I class (MHC).

16. each method of claim 1-11, wherein the mycobacterium polypeptide comprises 9 to 20 continuous amino acids of the shown amino acid sequence specificity of SEQ IDNO:61 in conjunction with the main histocompatibility complex of I class (MHC).

17. each method of claim 1-16, wherein biological sample is the CD3 of monocyte, sputum, lung biopsy sample, lymph node biopsy sample, saliva, cerebrospinal fluid or separation of peripheral blood lymphocytes, the separation of blood, separation ⁺The T cell.

18. each method of claim 1-16 is wherein with CD8 ⁺The T cell is in external and mycobacterium polypeptide co-incubation.

19. each method of claim 1-18, it also comprises the delayed allergy of detection at Much's bacillus.

20. each method of claim 1-19, it also is included in from the existence that detects the polynucleotide of mycobacterium polypeptide or coded polypeptide in the sample of object, and wherein the mycobacterium polypeptide comprises SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ IDNO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or the amino acid sequence shown in one of the shown amino acid sequence of one of SEQ ID NO:39-83.

21. the method for claim 19, it comprises the existence that detects the mycobacterium polypeptide.

22. comprising, the method for claim 21, the existence that wherein detects the mycobacterium polypeptide use the antibody of specificity in conjunction with the mycobacterium polypeptide.

23. the method for claim 20, it comprises the existence that detects polynucleotide.

24. the method for claim 23 determines that wherein the existence of polynucleotide comprises the use polymerase chain reaction.

25. specificity is in conjunction with the method for the T cell of mycobacterium polypeptide expression CD8 in the detected object, wherein to liking children, doubtful object or doubtful object with the outer m tuberculosis infection of lung with latent tuberculosis mycobacterial infections, described method comprises (A) and will contact with reagent from the peripheral blood lymphocytes that object is separated to, and described reagent comprises

(1) mycobacterium polypeptide, it comprises SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ IDNO:11 or SEQ ID NO:12, SEQ ID NO:39, at least 9 to 20 continuous amino acid of SEQ ID NO:61 or any at least one shown amino acid sequence of SEQ IDNO:39-83, wherein said 9 to 20 continuous amino acid specificitys are in conjunction with the main histocompatibility complex of I class (MHC);

(2) HLA heavy chain polypeptide and B2M; And

(3) streptavidin, wherein reagent is mark or unlabelled; And

(B) detect the existence of the reagent combine with peripheral blood lymphocytes, detection specificity is in conjunction with the T cell of mycobacterium polypeptide expression CD8 thus.

26. the method for claim 25, it also comprises the quantity of the CD8+T cell of binding reagents is carried out quantitatively.

27. the method for claim 25, wherein reagent is mark.

28. the method for claim 25, wherein reagent fluorophore mark.

29. the method for claim 25, wherein to as if children, and wherein detect specificity and show that in conjunction with the T cell of mycobacterium polypeptide expression CD8 described children suffer from pulmonary tuberculosis.

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